DENTAL  DEPARTMENT 

Bift  of 
Dr.  Henry  ?/.  V.'oirick 


Culture  of  glanders  bacillus  upon  cooked  potato  (Loftier) 


A  TEXT-BOOK 


UPON  THE 


PATHOGENIC  BACTERIA 


FOR 


STUDENTS  OF  MEDICINE  AND  PHYSICIANS 


JOSEPH  McFARLAND,  M.D. 


Professor  of  Pathology  in  the  Medico-Chirurgical  College,  Philadelphia;  Pathologist  to 
the  Medico-Chirurgical  Hospital  and  to  the  Rush  Hospital  for  Consump- 
tion and  Allied  Diseases,  Philadelphia;  Fellow  of  the 
College  of  Physicians  of  Philadelphia. 


WITH    134    ILLUSTRATIONS 


SECOND  EDITION,  REVISED  AND  ENLARGED 


PHILADELPHIA 

W.    B.    SAUNDERS 

925   WALNUT    STREET 
1898 


Copyright,  1898    by 
W.     B.    SAUNDERS 


ttlCTROtYPIO 


. 

\       V  PRESS    OF 

1  t       W     B.   SAUNOERS,    PHILAD/ 


TO 
MY  HONORED  AND  BELOVED  GRANDFATHER 

MR.  JACOB   GRIM 

WHOSE   PARENTAL   LOVE   AND    LIBERALITY   HAVE   ENABLED   ME  TO   PURSUE 
MY    MEDICAL    EDUCATION 

THIS    BOOK    IS    AFFECTIONATELY    DEDICATED 


PREFACE  TO  THE  SECOND  EDITION. 


IN  preparing  this  second  edition  of  u  Pathogenic  Bac- 
teria ' '  I  have  endeavored  to  bring  the  work  up  to  date 
in  all  departments  of  the  subject  by  introducing  brief 
mention  of  all  recent  work  accomplished  in  bacteriology. 
In  order  to  aid  the  student  whose  particular  interest 
might  make  him  desire  to  refer  to  the  original  papers, 
I  have  thought  well  to  depart  from  the  plan  of  the  orig- 
inal work  and  give  the  references  in  the  form  of  foot- 
notes. 

Without  departing  too  much  from  the  primary  descrip- 
tive purpose  of  the  book,  I  have  made  it  a  special  point 
to  add  considerably  to  the  amount  of  technique  it  con- 
tains, and  so  make  it  fulfill  the  double  purpose  of  a  sys- 
tematic work  upon  bacteria  and  a  laboratory  guide. 

New  chapters  have  been  added  dealing  with  the  bac- 
teriology of  Whooping-cough,  Mumps,  Yellow  Fever, 
Hog-cholera,  and  Swine-plague  ;  describing  the  Bacillus 
aerogenes  capsulatus  and  the  Proteus  vulgaris  ;  and 
describing  the  Methods  of  Determining  the  Value  of 
Antiseptics  and  Germicides,  and  of  Determining  the 
Thermal  Death-point. 

To  a  number  of  friendly  readers  whose  suggestions 
have  been  helpful  in  improving  the  work,  I  desire  to 
extend  my  sincere  thanks. 

JOSEPH   McFARLAND 


PREFACE. 


THE  following  pages  are  intended  to  convey  to  the 
reader  a  concise  account  of  the  technical  procedures 
necessary  in  the  study  of  bacteriology,  a  brief  descrip- 
tion of  the  life-history  of  the  important  pathogenic 
bacteria,  and  sufficient  description  of  the  pathological 
lesions  accompanying  the  micro-organismal  invasions 
to  give  an  idea  of  the  origin  of  symptoms  and  the 
causes  of  death. 

The  work  being  upon  Pathogenic  Bacteria,  it  does 
not  cover  the  whole  scope  of  parasitology,  and  the 
parasites  of  higher  orders  are  all  omitted.  Malaria  and 
amebic  dysentery  are  omitted  as  logically  as  tape-worms 
and  pediculi.  The  higher  fungi  are  also  omitted,  both 
because  they  are  not  bacteria  and  because  their  proper 
consideration  would  make  a  small  book  in  itself. 

In  leaving  out  the  non-pathogenic  bacteria  of  course 
a  stumbling-block  was  encountered.  The  Sarcina  ven- 
triculi,  for  instance,  may  be  a  cause  of  dyspepsia,  yet 
can  scarcely  be  regarded  as  pathogenic,  and,  together 
with  other  similar  bacteria  of  questionable  deleterious 
operation,  has  been  omitted  ;  on  the  other  hand,  it 
has  been  thought  advisable  to  include  and  describe 
somewhat  at  length  a  long  list  of  spirilla  similar  to, 
and  probably  closely  allied  with,  the  spirillum  of 
cholera,  yet  not  the  cause  of  any  particular  diseased 
condition. 


10  WEFACE. 

The  aim  has  been  to  describe  only  such  bacteria  as 
can  be  proven  pathogenic  by  the  lesions  or  toxins 
which  they  engender,  and,  while  considering  them,  to 
mention  as  fully  as  is  necessary  the  species  with  which 
they  may  be  confounded. 

The  book,  of  course,  will  find  its  proper  sphere  of 
usefulness  in  the  hands  of  medical  students  ;  its  pages, 
however,  will  be  found  to  contain  much  that  will  be 
of  interest  and  profit  to  those  practitioners  of  medicine 
who  graduated  before  modern  science  had  thrown  its 
lijjht  upon  the  etiology  of  disease. 

In  writing  this  work  the  popular  text-books  have 
been  drawn  upon.  Hiippe,  Fliigge,  Sternberg,  Frankel, 
(jutither,  Thoinot  and  Masselin,  and  others  have  been 
freely  consulted. 

The  illustrations  are  mainly  reproductions  of  the  best 
the  world  affords,  and,  being  taken  from  the  great  stand- 
ards, are  surely  superior  to  anything  new  covering  the 
same  ground.  Credit  has  carefully  been  given  for  each 
illustration. 

J.McF. 
,  Feb.  i,  1896. 


CONTENTS. 


PART  I.    GENERAL  CONSIDERATIONS. 

PAGE 

INTRODUCTION ' 17 

CHAPTER  I. 
BACTERIA 30 

CHAPTER  II. 
THE  BIOLOGY  OF  BACTERIA 43 

CHAPTER  III. 
IMMUNITY  AND  SUSCEPTIBILITY 65 

CHAPTER  IV. 
METHODS  OF  OBSERVING  BACTERIA 86 

CHAPTER  V. 
STERILIZATION  AND  DISINFECTION 105 

CHAPTER  VI. 
THE  CULTIVATION  OF  BACTERIA  ;  CULTURE-MEDIA 124 

CHAPTER  VII. 
CULTURES,  AND  THEIR  STUDY 139 

CHAPTER  VIII. 
THE  CULTIVATION  OF  ANAEROBIC  BACTERIA 153 

CHAPTER  IX. 

EXPERIMENTATION  UPON  ANIMALS 158 

11 


12  CONTENTS. 

CHAPTER  X. 

PAGE 

THE  RECOGNITION  OF  BACTERIA 163 

CHAPTER  XI. 
THE  BACTERIOLOGIC  EXAMINATION  OF  AIR 164 

CHAPTER  XII. 
THE  BACTERIOLOGIC  EXAMINATION  OF  WATER 169 

CHAPTER  XIII. 
Tin:  BACTERIOLOGIC  EXAMINATION  OF  SOIL 174 

CHAPTER   XIV. 

Tin.  Tin-:  KM  A  i,   DEATH-POINT  AND  THE  VALUE  OF  GERMI- 
CIDES     176 


PART   II.    SPECIFIC    DISEASES    AND    THEIR 
BACTERIA. 


A.  THE  PHLOGISTIC  DISEASES. 


I.  THE  ACUTE  INFLAMMATORY  DISEASES. 


CHAPTER  I. 

Si   l-IM   K  \TIOX  ;    r.ONOKRHK  A  \    MUMPS  .  .    lS2 


II.  THE  CHRONIC  INFLAMMATORY  DISEASES. 


CHAPTER  I. 

Tri'.i-iKcn.osis ->()x 

CHAPTER  II. 

LEPROSY 241 

CHAPTER  III. 

.  24S 


CONTENTS.  13 

CHAPTER  IV. 

PAGE 

SYPHILIS 255 

CHAPTER  V. 

ACTINOMYCOSIS 260 

CHAPTER  VI. 
MYCETOMA,  OR  MADURA- FOOT 266 

CHAPTER  VII. 
FARCIN  DU  BCEUF 270 

CHAPTER  VIII. 
RHINOSCLEROMA 273 


B.   THE   TOXIC    DISEASES. 


CHAPTER  I. 
TETANUS     274 

CHAPTER  II. 
DIPHTHERIA 284 

CHAPTER   III. 
HYDROPHOBIA,  OR  RABIES 306 

CHAPTER    IV. 

CHOLERA   AND   SPIRILLA    RESEMBLING  THE    CHOLERA  SPI- 
RILLUM   311 

CHAPTER  V. 
PNEUMONIA 345 


I4  CONTENTS 


C  THE  SEPTIC  DISEASES. 


CHAPTER  I- 
ANTHRAX    

CHAPTER  II. 

...    .366 


TYPHOID  FEVER  ......... 

CHAPTER  HI. 
THE  BACILLUS  COLI  COMMUNIS 


CHAPTER  IV. 
Yr.u.ow  FEVER   .................... 

CHAPTER   V. 

OlKKI.N-CHOLERA   .....................  4°9 


CHAPTER  VI. 
HOG-CHOLERA 


CHAPTER   VII. 
SWIM    i  i.AGUE  .......................  42° 

CHAPTER   VIII. 

TVI'HI  s     Ml   Hlt'M   ......................  423 

CHAPTER    IX. 
'  PTH  i  MIA  ...  .............  426 

CH  \1TER    X. 
\I-SIN..    1-RVBR  .....  ............  431 


Til  M'TI-R     XI. 

PLAOUI     .  .      .  . 


CHAI'TI-R    XII. 

.....  •    •  443 


CONTENTS.  15 

CHAPTER   XIII. 

PAGE 

INFLUENZA     446 

CHAPTER  XIV. 
MEASLES 451 


D.  MISCELLANEOUS. 


CHAPTER  I. 
SYMPTOMATIC  ANTHRAX 453 

CHAPTER  II. 
MALIGNANT  EDEMA ". 459 

CHAPTER   III. 
THE  BACILLUS  AEROGENES  CAPSULATUS 463 

CHAPTER  IV. 
THE  BACILLUS  PROTEUS  VULGARIS     472 

CHAPTER  V. 
WHOOPING-COUGH 476 


INDEX 481 


PATHOGENIC    BACTERIA. 


PART  I.     GENERAL   CONSIDERATIONS. 


INTRODUCTION. 

IT  is  incorrect  to  begin  the  consideration  of  bacteriol- 
ogy, as  is  so  often  done,  with  the  probable  discoverer  of 
bacteria,  Leeuwenhoek,  or  with  the  so-called  "Father  of 
bacteriology, ' '  Henle.  The  controversies  and  ideas  which 
stimulated  the  investigations  and  researches  which  have 
brought  us  to  our  present  state  of  knowledge  were  begun 
hundreds  of  years  before  the  beginning  of  the  Christian  era. 

Excepting  such  as  taught  and  believed  that  "in  six 
days  the  Lord  made  heaven  and  earth,  the  sea  and  all 
that  in  them  is,"  or  a  kindred  theory  of  the  origin  of 
things,  the  thinkers  of  antiquity  never  seem  to  have 
doubted  that  under  favorable  conditions  life,  both  animal 
and  vegetable,  might  arise  spontaneously. 

Among  the  early  Greeks  we  find  that  Anaximander 
(43d  Olympiad,  610  B.  c.)  of  Miletus  held  the  theory  that 
animals  were  formed  from  moisture.  Empedocles  of 
Agrigentum  (450  B.  c.)  attributed  to  spontaneous  genera- 
tion all  the  living  beings  which  he  found  peopling  the 
earth.  Aristotle  (B.  c.  384)  is  not  so  general  in  his  view 
of  the  subject,  but  asserts  that  "sometimes  animals  are 
formed  in  putrefying  soil,  sometimes  in  plants,  and  some- 
times in  the  fluids  of  other  animals. ' '  He  also  formulated 
a  principle  that  "every  dry  substance  which  becomes 
moist,  and  every  moist  body  which  becomes  dried,  pro- 
duces living  creatures,  provided  it  is  fit  to  nourish  them." 

2  17 


l8  /'.  /  TH(  ">(,EMC  BACTERIA. 

Three  centuries  later,  in  his  disquisition  upon  the 
Pythagorean  philosophy,  we  find  Ovid  defending  the 
same  doctrine:1 

this  sure  experiment  we  know 
That  living  creatures  from  corruption  grow  : 
Hide  in  a  hollow  pit  a  slaughter'd  steer, 
Bees  from  his  putrid  bowels  will  appear, 
Who,  like  their  parents,  haunt  the  fields  and  bring 
Their  honey-harvest  home,  and  hope  another  spring 
The  warlike  steed  is  multiplied,  we  find, 
To  wasps  and  hornets  of  the  warrior  kind. 
Cut  from  a  crab  his  crooked  claws,  and  hide 
The  rest  in  earth,  a  scorpion  thence  will  glide, 
And  shoot  his  sting  ;  his  tail  in  circles  toss'd 
Refers  the  limbs  his  backward  father  lost  ; 
And  worms  that  stretch  on  leaves  their  filmy  loom 
Crawl  from  their  bags  and  butterflies  become. 
The  slime  begets  the  frog's  loquacious  race  ; 
Short  of  their  feet  at  first,  in  little  space, 
With  arms  and  legs  endued,  long  leaps  they  take, 
Raised  on  their  hinder  part,  and  swim  the  lake, 
And  waves  repel  ;  for  nature  gives  their  kind, 
To  that  intent,  a  length  of  legs  behind." 

Not  only  was  the  doctrine  of  spontaneous  generation 
of  life  current  among  the  ancients,  but  we  find  it  persist- 
ing through  the  Middle  Ages,  and  descending  to  our  own 
generation  to  be  an  accidental  but  important  factor  in 
the  development  of  a  new  branch  of  science.  In  1542, 
in  his  treatise  called  DC  Subtilitatc,  we  find  Cardan  as- 
serting that  water  engenders  fishes,  and  that  many  ani- 
mals spring  from  fermentation.  Van  Hclmont  gives 
special  instructions  for  the  artificial  production  of  mice, 
and  Kircher  in  his  Mninins  Subtaranciis  (chapter  4lDe 
;»ermiu  Rernm  ")  describes  and  actually  figures  cer- 
tain animals  which  were  produced  under  his  own  eyes 
by  the  transforming  influence  of  water  on  fragments  of 
from  different  plants.2 


1  i  hrid'l  -l/  '••"    '.*;!->sss,  translate:  U  Mr.  Dryden,  published  by  Sir  Samuel 
Garth.  1794. 

1  See  Tyndall  :  Floating  Matter  in  tht  Air. 


INTRODUCTION.  19 

About  1668,  Francesco  Redi  seems  to  have  been  the 
first  to  doubt  that  the  maggots  familiar  in  putrid  meat 
arose  de  novo  :  "Watching  meat  in  its  passage  from 
freshness  to  decay,  prior  to  the  appearance  of  maggots, 
he  invariably  observed  flies  buzzing  around  the  meat  and 
frequently  alighting  on  it.  The  maggots,  he  thought, 
might  be  the  half-developed  progeny  of  these  flies. 
Placing  fresh  meat  in  a  jar  covered  with  paper,  he  found 
that  although  the  meat  putrefied  in  the  ordinary  way, 
it  never  bred  maggots,  while  meat  in  open  jars  soon 
swarmed  with  these  organisms.  For  the  paper  he  sub- 
stituted fine  wire  gauze,  through  which  the  odor  of.  the 
meat  could  rise.  Over  it  the  flies  buzzed,  and  on  it  they 
laid  their  eggs,  but  the  meshes  being  too  small  to  per- 
mit the  eggs  to  fall  through,  no  maggots  generated  in 
the  meat;  they  were,  on  the  contrary,  hatched  on  the 
gauze.  By  a  series  of  such  experiments  Redi  destroyed 
the  belief  in  the  spontaneous  generation  of  maggots  in 
meat,  and  with  it  many  related  beliefs." 

It  was  not  long  before  Leeuwenhoek,  Vallismeri, 
Swammerdan,  and  others,  following  the  trend  of  Redi's 
work,  contributed  additional  facts  in  favor  of  his  view, 
and  it  may  safely  be  asserted  that  ever  since  the  time 
of  this  eminent  man  the  tide  of  scientific  opinion  has 
turned  more  and  more  strongly  against  the  idea  that 
life  is  spontaneously  generated. 

About  this  time  (1675)  one  whose  name  has  been 
already  mentioned,  Anthony  van  Leeuwenhoek,  and  who 
is  justly  called  the  "Father  of  microscopy,"  came  into 
prominence.  An  optician  by  trade,  Leeuwenhoek  devoted 
much  time  to  the  perfection  of  the  compound  micro- 
scope, which  was  just  coming  into  use.  The  science  of 
optics,  however,  was  not  sufficiently  developed  to  enable 
him  to  overcome  the  errors  of  refraction,  and  after  the 
loss  of  much  time  he  turned  to  the  simple  lens,  using  it 
in  so  careful  and  remarkable  a  manner  as  to  be  able 
to  record  his  observations  in  one  hundred  and  twelve 
contributions  to  the  Philosophical  Transactions.  Leeu- 


20  PATHOGENIC  BACTERIA. 

wenhoek,  among  other  things,  demonstrated  the  conti- 
nuity of  arteries  and  veins  through  intervening  capil- 
s,  thus  affording  ocular  proof  of  Harvey's  discovery 
of  the  circulation  of  the  blood;  discovered  the  rotifers, 
and  also  the  bacteria,  seeing  them  first  in  saliva. 

Although  one  of  those  who  contributed  to  the  support 
of  Redi's  arguments  against  the  spontaneous  generation 
of  maggots,  Leeuwenhoek  involuntarily  reopened  the  old 
controversy  about  spontaneous  generation  by  bringing 
forward  a  new  world,  peopled  by  creatures  of  such  ex- 
treme minuteness  as  to  suggest  not  only  a  close  relation- 
ship to  the  ultimate  molecules  of  matter,  but  an  easy 
transition  from  them.  Interested  in  Leeuwenhoek' s 
work,  Plencig  of  Vienna  became  convinced  that  there 
was  an  undoubted  connection  between  the  microscopic 
animals  exhibited  by  the  microscope  and  the  origin  of 
disease,  and  advanced  this  opinion  as  early  as  1762. 
Unfortunately,  the  opinions  of  Plencig  seem  not  to  have 
been  accepted  by  others,  and  were  soon  forgotten. 

In  succeeding  years  the  development  of  the  compound 

microscope  showed  these  minute  organisms  to  exist  in 

such  numbers  that  putrescent  infusions,  both  animal  and 

table,  literally  teemed  with  them,  one  drop  of  such 

a  liquid  furnishing  a  banquet  for  millions. 

Much  hostility  arose  in  the  scientific  world  as  years 
went  on  until  two  schools  attained  prominence — one 
headed  by  Buffon,  whose  doctrine  was  that  of  "organic 
molecules;"  the  other  championed  by  Needham,  whose 
doctrine  was  the  existence  of  a  "vegetative  force"  which 
drew  the  molecules  together. 

Experimentation  was  begun  and  attracted  much  atten- 
Aniong  the  pioneers  was  Abbe  Lazzaro  Spallan- 
/.mi  1777),  who  filled  flasks  with  organic  infusions, 
sealed  their  necks,  and,  after  subjecting  their  contents 
to  the  temperature  of  boiling  water,  placed  them  under 
conditions  favorable  for  the  development  of  life,  without, 
however,  being  able  to  produce  it.  Spallan/ani's  critics, 
however,  objected  to  his  experiment  on  the  ground  that 


INTRODUCTION.  21 

air  is  essential  to  life,  and  that  in  his  flasks  the  air  was 
excluded  by  the  hermetically-sealed  necks. 

Schulze  (1836)  set  the  objection  aside  by  filling  a  flask 
only  half  full  of  distilled  water,  to  which  animal  and 
vegetable  matters  were  added,  boiling  the  contents  to 
destroy  the  vitality  of  any  organisms  which  might  al- 
ready exist  in  them,  then  sucking  daily  into  the  flask  a 
certain  amount  of  air  which  had  passed  through  a  series 
of  bulbs  containing  concentrated  sulphuric  acid,  in  which 
it  was  supposed  that  whatever  germs  of  life  the  air  might 
contain  would  be  destroyed.  This  flask  was  kept  from 
May  to  August;  air  was  passed  through  it  daily,  yet  with- 
out the  development  of  any  infusorial  life. 

The  term  "infusorial  life"  having  been  used,  here  it 
is  well  to  observe  that  during  all  the  early  part  of  their 
recognized  existence  the  bacteria  were  regarded  as  ani- 
mal organisms  and  classed  among  the  infusoria. 

Cagniard  Latour  and  Schwann  in  the  year  1837  suc- 
ceeded in  proving  that  the  minute  oval  bodies  which  had 
been  observed  in  yeast  since  the  the  time  of  Leeuwenhoek 
were  living  organisms — vegetable  forms — capable  of 
growth;  and  when  Boehm  succeeded  a  year  later  in  de- 
monstrating their  occurrence  in  the  stools  of  cholera,  and 
conjectured  that  the  process  of  fermentation  was  con- 
cerned in  the  causation  of  that  disease,  the  study  of  these 
low  forms  of  life  received  an  immense  impetus  from  the 
important  position  which  they  began  to  assume  in  rela- 
tion to  medical  science. 

The  experiments  of  Schwann,  by  proving  that  the 
free  admission  of  calcined  air  to  closed  vessels  contain- 
ing putrescible  infusions  was  without  effect,  while  the 
admission  of  ordinary  air  brought  about  decomposition, 
suggested  that  the  causes  of  putrefaction  which  were  in 
the  air  were  living  entities. 

In  1862,  Pasteur  published  a  paper  "  On  the  Organized 
Corpuscles  existing  in  the  Atmosphere,"  in  which  he 
showed  that  many  of  the  floating  particles  which  he 
had  been  able  to  collect  from  the  atmosphere  of  his 


22  PATHOGENIC  BACTERIA. 

laboratory  were  organized  bodies.  If  these  were  planted 
in  sterile  infusions,  abundant  crops  of  micro-organisms 
were  obtainable.  By  the  use  of  more  refined  methods 
he  repeated  the  experiments  of  Schwann  and  others,  and 
showed  clearly  that  u  the  cause  which  communicated  life 
to  his  infusions  came  from  the  air,  but  was  not  evenly  dis- 
tributed through  it." 

Three  years  later  he  showed  that  the  organized  cor- 
puscles which  he  had  found  in  the  air  were  the  spores  or 
seeds  of  minute  plants,  and  that  many  of  them  possessed 
the  property  of  withstanding  the  temperature  of  boiling 
water — a  property  which  explained  the  peculiar  results 
of  many  previous  experimenters,  who  failed  to  prevent 
the  development  of  life  in  boiled  liquids  enclosed  in 
hermetically-sealed  flasks. 

Chevreul  and  Pasteur  (about  1836)  proved  that  animal 
solids  did  not  putrefy  or  decompose  if  kept  free  from 
the  access  of  germs,  and  thus  suggested  to  surgeons  that 
the  putrefaction  which  occurred  in  wounds  was  due  rather 
to  the  entrance  of  something  from  without  than  to  some 
change  within.  The  deadly  nature  of  the  discharges 
from  these  wounds  had  been  shown  in  a  rough  manner 
by  Gaspard  as  early  as  1822  by  injecting  some  of  the 
material  into  the  veins  of  animals. 

Examinations  of  the  blood  of  diseased  animals  were 
now  begun,  and  Pol  lender  (1849)  and  Davaine  (1850) 
succeeded  in  demonstrating  the  presence  of  the  anthrax 
bacillus  in  that  disease.  Several  years  later  (1863)  Da- 
vaine,  having  made  numerous  inoculation-experiments, 
demonstrated  that  this  bacillus  was  the  tnatcrics  morbi 
of  the  disease. 

Tvndall  enlarged  upon   the  experiments  of  Pasteur, 

and  very  conclusively  proved  that  the  micro-organismal 

>  were  in  the  dust  suspended  in  the  atmosphere,  not 

ubiquitous  in  their  distribution.     His  experiments  were 

ingenious  and  are  of  interest  to  medical  men.     First 

niu-  lioht  wooden  chambers,  with  one  large  glass 

window   in    the  front  and  one  smaller  window  in  each 


INTRODUCTION.  23 

side,  he  arranged  a  series  of  empty  test-tubes  in  the 
bottom  and  a  pipette  in  the  top,  so  that  when  desired 
the  tubes,  one  by  one,  could  be  filled  through  it.  The 
chamber  was  first  submitted  to  an  optical  test  to  deter- 
mine the  purity  of  its  atmosphere,  and  was  allowed  to 
stand  undisturbed  and  unused  until  a  powerful  ray  of 
light  passed  through  the  side  windows  failed  to  reflect 
rays  from  suspended  particles  of  dust  when  viewed  from 
the  front.  When  the  dust  had  settled  so  as  to  allow  the 
optical  test  of  its  purity,  the  tubes  were  filled  with  urine, 
beef-broth,  and  a  variety  of  animal  and  vegetable  broths, 
boiled  by  submergence  in  a  pan  of  hot  brine;  the  tubes 
were  then  allowed  to  remain  undisturbed  for  days,  weeks, 
or  months.  In  nearly  every  case  life  failed  to  develop 
after  the  purity  of  the  atmosphere  was  established. 

In  1873,  Obermeier  observed  that  actively  motile,  flex- 
ible spiral  organisms  were  present  in  large  numbers  in 
the  blood  of  patients  in  the  febrile  stages  of  relapsing 
fever. 

Thus  evidence  slowly  accumulated  to  establish  the 
theory  for  which  Henle  had  labored  as  early  as  1821,  that 
for  many  diseases  at  least  there  was  a  distinct  and  specific 
contagium  vivum,  and  the  UGERM  THEORY"  was  pro- 
pounded. 

Is  it  not  strange  that  the  very  idea  which  was  to  be  the 
outcome  of  all  this  investigation  and  discussion — an  idea 
which  would  form  a  new  era  in  scientific  medicine  and 
become  a  fundamental  principle  of  pathology — was  one 
which  had  been  conceived  and  taught  by  a  philosopher 
who  lived  nearly  two  thousand  years  ago?  Among  the 
numerous  works  of  Varro l  is  one  entitled  Rerum  Rusti- 
cantm  libri  tres,  from  which  the  following  is  quoted : 
"Animadvertendum  etiam,  si  qua  erunt  loca  palustria— 
quod  crescunt  animalia  quaedam  minuta,  quae  non  pos- 
sunt  oculi  consequi  et  per  ae'ra  intus  in  corpus  per  os  ac 
nares  perveniunt  atque  efficiunt  difficilis  morbus "  (L, 
xii.  2).—"  It  is  also  to  be  noticed,  if  there  be  any  marshy 

1  Univ.  Med.  Mag.,  vol.  iii.,  No.  3,  Dec.,  1890,  p.  152. 


24  PATHOGENIC  BACTERIA. 

places,  that  certain  minute  animals  breed  [there]  which 
are  invisible  to  the  eye,  and  yet,  getting  into  the  sys- 
tem through  mouth  and  nostrils,  cause  serious  disor- 
ders (diseases  which  are  difficult  to  treat) "-a  doctrine 
which,  as  Prof.  Lamberton,  to  whom  the  writer  is  in- 
debted for  the  extract,  points  out,  is  handed  down  to  us 
from  uthe  days  of  Cicero  and  Caesar,"  yet  corresponds 
closely  to  the  ideas  of  malaria  which  we  entertain  at 
present. 

Pasteur  had  long  before  suggested  that  for  the  different 
kinds  of  fermentation  there  must  be  specific  ferments, 
and  by  fractional  cultures  had  succeeded  in  roughly  sepa- 
rating them. 

Klebs,  who  was  one  of  the  pioneers  of  the  germ 
theory,  published  in  1872  his  work  upon  septicemia  and 
iia,  in  which  he  expressed  himself  convinced  that 
the  causes  of  these  diseases  must  come  from  without  the 
body.  Billroth  strongly  opposed  such  an  idea,  asserting 
that  fungi  had  no  especial  importance  either  in  the  pro- 
cesses of  disease  or  in  those  of  decomposition,  but  that, 
•intf  everywhere  in  the  air,  they  rapidly  developed  in 
the  body  as  soon  as  through  putrefaction  a  4 '  Faulniss- 
/vmoid,"  or  through  inflammation  a  u  phlogistische- 
/ A  moid,"  supplying  the  necessary  feeding-grounds,  was 
produced. 

Klebs  was  not  alone  in  the  opposition  aroused.  Da- 
vaine  no  sooner  announced  the  contagium  of  anthrax 
than  critics  declared  that  inasmuch  as  he  introduced 
blood  from  the  diseased  animal  into  the  other  animal 
to  whom  the  disease  was  to  be  communicated,  it  was 
altogether  unreasonable  to  believe  the  bacilli  which  were 
in  all  probability  accidentally  present  in  that  blood  were 
the  cause  of  the  disease. 

In  1875  the  number  of  scientific  men  who  had  embraced 
the  nerm  theory  of  disease  was  small,  and  most  of  those 
who  accepted  it  were  experimenters.  A  great  majority 
of  medical  men  either  believed,  like  Billroth,  that  the 
presence  of  fun-i  where  decomposition  was  in  progress 


INTRODUCTION.  25 

was  an  accidental  result  of  their  universal  distribution, 
or,  being  still  more  conservative,  retained  the  old  un- 
questioning faith  that  the  bacteria,  whose  presence  in 
putrescent  wounds  as  well  as  in  artificially  prepared 
media  was  unquestionable,  were  spontaneously  generated 
there. 

The  following  extracts  from  Tyndall's  work1  will  illus- 
trate the  slow  growth  of  the  germ  theory  even  among 
men  of  eminence  : 

u  At  a  meeting  of  the  Pathological  Society  of  London, 
held  April  6,  1875,  the  '  germ  theory '  of  disease  was 
formally  introduced  as  a  subject  for  discussion,  the  debate 
being  continued  with  great  ability  and  earnestness  at  sub- 
sequent meetings.  The  conference  was  attended  by 
many  distinguished  medical  men,  some  of  whom  were 
profoundly  influenced  by  the  arguments,  and  none  of 
-whom  disputed  the  facts  brought  forward  against  the 
theory  on  that  occasion." 

'The  leader  of  the  debate,  and  the  most  prominent 
speaker,  was  Dr.  Bastian,  to  whom  also  fell  the  task  of 
replying  on  all  the  questions  raised." 

* '  The  coexistence  of  bacteria  and  contagious  disease 
was  admitted ;  but,  instead  of  considering  these  organisms 
as  probably  the  essence,  or  an  inseparable  part  of  the  es- 
sence, of  the  contagium,  Dr.  Bastian  contended  that  they 
were  pathological  products  spontaneously  generated  in  the 
body  after  it  had  been  rendered  diseased  by  the  real  con- 
tagium. ' ' 

"The  grouping  of  the  ultimate  particles  of  matter  to 
form  living  organisms  Dr.  Bastian  considered  to  be  an 
operation  as  little  requiring  the  action  of  antecedent  life 
as  their  grouping  to  form  any  of  the  less  complex  chem- 
ical compounds. "  u  Such  a  position  must,  of  course, 
stand  or  fall  by  the  evidence  which  its  supporter  is  able 
to  produce,  and  accordingly  Dr.  Bastian  appeals  to  the 
law  and  testimony  of  experiment  as  demonstrating  the 
soundness  of  his  view."  "  He  seems  quite  aware  of  the 

1  Op.  dt. 


26  PATHOGENIC  BACTERIA. 

gravity  of  the  matter  at  hand  ;  this  is  his  deliberate  and 
almost  solemn  appeal  :  k  With  the  view  of  settling  these 
•ions,  therefore,  we  may  carefully  prepare  an  infusion 
from  some  animal  tissue,  be  it  muscle,  kidney,  or  liver  ; 
we  may  place  it  in  a  flask  whose  neck  is  drawn  out 
and  narrowed  in  the  blowpipe  flame;  we  may  boil  the 
fluid,  seal  the  vessel  during  ebullition,  and,  keeping  it 
in  a  warm  place,  may  await  the  result,  as  I  have  often 

done After  a  variable  time  the  previously  heated 

fluid  within  the  hermetically-sealed  flask  swarms  more 
or  less  plentifully  with  bacteria  and  allied  organisms, 
even  though  the  fluids  have  been  so  much  degraded  in 
quality  by  exposure  to  the  temperature  of  212°  F.,  and 
have  in  all  probability  been  rendered  far  less  prone  to 
engender  independent  living  units  than  the  unheated 
fluids  in  the  tissues  would  be.'  ' 

These  somewhat  lengthy  quotations  are  of  great  in- 
terest, for  they  show  exactly  the  state  of  the  scientific 
mind  at  a  period  as  recent  as  twenty  years  ago. 

In  1877  the  introduction  of  the  anilin  dyes  by  Weigert 
made  possible  a  much  more  thorough  investigation  of 
the  bacteria  by  enabling  the  observers  to  color  them 
intensely,  and  thus  detect  their  presence  in  tissues  and 
organs  where  their  transparency  had  caused  them  to  be 
overlooked. 

Rapid  strides  were  immediately  made,  and  before 
another  decade  had  passed  discoveries  were  so  numerous 
and  convincing  that  it  was  impossible  to  doubt  that  bac- 
teria were  causes  of  disease. 

Before  the  publication  of  the  discoveries  of  which  we 
speak,  however,  there  was  suj^ested  a  practical  applica- 
tion of  the  little  known  about  bacteria  which  produced 
greater  agitation  and   incited  more  observation  and  ex- 
perimentation than  am  thin-  su.i^ested  in  surgery  since 
tlu-   introduction  of  anesthetics— namely,  antisepsis. 
4  The  seminal   thought  of  antiseptic  surgery  may  per- 
ed  to  John  Colbach,  a  member  of  the  College 
of  Physicians,  England,  whose  collection  of  tracts,  printed 


INTRODUCTION.  27 

1704,  contained  a  description  of  a  new  and  secret  method 
of  treating  wounds,  by  which  healing  took  place  quickly 
without  inflammation  or  suppuration;  but  it  is  to  one  of 
old  Scotia's  sons,  Sir  Joseph  Lister,  that  the  everlasting 
gratitude  of  the  world  is  due  for  the  knowledge  we  pos- 
sess in  regard  to  the  relation  existing  between  micro- 
organisms and  inflammation  and  suppuration,  and  the 
power  to  render  wounds  aseptic  through  the  action  of 
germicidal  substances."  l 

Lister  was  not  the  discoverer  of  carbolic  acid  nor  of 
the  fact  that  it  would  kill  bacteria;  but,  convinced  that 
inflammation  and  suppuration  were  due  to  the  entrance 
of  germs  from  the  air,  instruments,  fingers,  etc.  into 
wounds,  he  suggested  the  antisepsis  which  would  insist 
upon  the  use  of  sterile  instruments  and  clean  hands  and 
towels;  which  would  keep  the  surface  of  the  wound 
moist  with  a  germicidal  solution  to  kill  such  germs  as 
accidentally  entered;  and  which  would  conclude  an  ope- 
ration by  a  protective  dressing  to  exclude  the  entrance  of 
germs  at  a  subsequent  period. 

Listerism,  originated  (1875)  a  few  years  before  Koch 
published  his  famous  work  on  the  Wundinfectionskrank- 
heiten  (traumatic  infectious  diseases)  (1878),  spread  slowly 
at  first,  but  surely  in  the  end,  to  all  departments  of  sur- 
gery and  obstetrics. 

The  discovery  of  the  yeast-plant  by  Latour  and 
Schwann  as  the  cause  of  fermentation,  and  the  later  dis- 
covery by  Bassi  of  the  yeast-like  plant  causing  the  mias- 
matic contagious  disease  of  silkworms,  had  led  Henle 
(1840)  to  believe  that  the  cause  of  miasmatic,  infective, 
and  contagious  diseases  must  be  looked  for  in  fungi  or 
in  other  minute  living  organisms.  Unfortunately,  the 
methods  of  study  employed  in  Henle' s  time  prevented 
him  from  demonstrating  the  accuracy  of  his  belief. 

"  It  would  indeed  have  been  difficult  at  that  period  to 
satisfy  every  condition  that  he  required  to  be  fulfilled: 
the  methods  now  in  use  were  then  unknown,  and  have 

1  Agnew's  Surgery,  vol.  i.  chap.  ii. 


28  PATHOGENIC  BACTERIA. 

only  been  perfected  by  workers  as  it  has  been  found  nec- 
essary from  time  to  time  to  comply  in  the  most  minute 
detail  with  Henle's  conditions,  and  as,  one  point  being 
carried,  it  was  found  necessary  to  advance  on  others. 
The  first  of  these  was  that  a  specific  organism  should 
always  be  associated  with  the  disease  under  consideration. 
As  such  presence,  however,  might  be  accidental,  these 
organisms  were  not  only  to  be  found  in  pus,  etc.,  but  actu- 
ally in  the  living  body.  As  they  might  be,  even  then, 
merely  parasitic,  and  not  associated  directly  with  the 
causation  of  the  disease,  it  would  be  necessary  to  isolate 
the  germs,  the  contagium  organisms,  and  the  contagium 
fluids,  and  to  experiment  with  these  separately  with 
special  reference  to  their  power  of  producing  similar 
diseases  in  other  animals.  We  now  know  that  it  has 
only  been  by  strict  compliance  with  all  these  conditions, 
again  postulated  by  Koch,  that  the  most  brilliant  scien- 
tific observers  and  experimentalists  in  Germany,  France, 
England,  [and  America]  have  been  able  to  determine 
the  causal  connection  between  micro-organisms  and 
disease."  ! 

The  refined  methods  of  Pasteur,  but  more  especially 
of  Koch,  by  making  possible  the  fulfilment  of  the  pos- 
tulates of  Henle  caused  an  enormous  increase  in  the 
rapidity  with  which  data  upon  disease-germs  were  gath- 
ered. Almost  within  a  decade  the  causes  of  the  most 
important  specific  diseases  were  isolated  and  cultivated. 

In  1879,  Hausen  announced  the  discovery  of  bacilli  in 
the  cells  of  leprous  nodules.  The  same  year  Neisser 
discovered  the  gonococcus  to  be  specific  for  gonorrhea. 

In  1880  the  bacillus  of  typhoid  fever  was  first  observed 
by  Eberth,  and  independently  by  Koch. 

In  1880,  Pasteur  published  his  work  upon  "chicken- 
cholera.'1  In  tlu-  same  year  Sternberg  described  the 
pmnmococcus,  calling  it  the  micrococcus  Fasten n. 

In  i.ssj,  Koch  made  himself  immortal  by  his  discov- 
;<1  work  upon  the  tubercle  bacillus.  The  same 

llic.i.l  :    />',/< /,;m  and  their  Products,  p.  65. 


INTRODUCTION,  29 

year  Pasteur  published  a  work  upon  Rouget  du  Pore,  and 
L,6ffler  and  Schiitz  reported  the  discovery  of  the  bacillus 
of  glanders. 

In  1884,  Koch  reported  the  discovery  of  the  "comma 
bacillus,"  the  cause  of  cholera,  and  in  the  same  year 
Ivoffler  discovered  the  diphtheria  bacillus,  and  Nicolaier 
the  tetanus  bacillus. 

In  1892,  Canon  and  Pfeiffer  discovered  the  bacillus  of 
influenza. 

In  1892,  Canon  and  Pielicke  first  found  the  bacillus 
now  thought  to  be  specific  for  measles. 

In  1894,  Yersin  and  Kitasato  independently  isolated 
the  bacillus  causing  the  bubonic  plague  then  prevalent 
at  Hong-Kong. 

A  new  era  in  bacteriology,  and  probably  the  most 
triumphant  result  of  the  modern  scientific  study  of  dis- 
ease, was  inaugurated  in  1890  by  Behring,  who  presented 
to  the  world  the  ' '  Blood-serum  therapy, ' '  and  showed  as 
the  result  of  prolonged,  elaborate,  and  profound  study  of 
the  subject  of  immunity  that  in  the  blood  of  animals 
with  acquired  immunity  to  certain  diseases  (diphtheria 
and  tetanus)  a  substance  was  held  in  solution  which  was 
potent  to  save  the  lives  of  other  animals  suffering  from 
the  same  diseases. 


CHAPTER  I. 
BACTERIA. 

A  BACTERIUM  is  a  minute  vegetable  organism  consist- 
ing of  a  single  cell  principally  composed  of  an  albumin- 
ous substance,  which  Nencki  has  called  mycoprotein. 
Nencki  found  the  chemical  analysis  of  bacteria  in  the 
active  state  to  consist  of  82.42  per  cent,  of  water.  In 
ico  parts  of  the  dried  constituents  he  found  84. 20  parts 
of  mycoprotein;  6.04  of  fat;  4.72  of  ash;  5.04  of  unde- 
termined substances. 

Mycoprotein,  which  has  the  composition  C  52.32,  H 
7.55,  N  14.75,  is  a  perfectly  transparent,  generally  ho- 
mogeneous body,  which  probably  varies  somewhat  ac- 
cording to  the  species  from  which  it  is  obtained,  the 
culture-medium  in  which  it  is  grown,  and  the  vital 
products  which  the  organism  produces  by  its  growth. 
Sometimes  the  mycoprotein  is  granular,  as  in  bacillus 
megatherium  ;  sometimes  it  contains  fine  granules  of 
chlorophyl,  sulphur,  fat,  or  pigment.  Each  cell  is  sur- 
rounded by  a  cell-wall,  which  in  some  species  shows  the 
cellulose  reaction  with  iodin. 

When  subjected  to  the  influence  of  nuclear  stains  the 
bacteria  not  only  take  the  stain  faintly,  but  in  such  a 
manner  as  to  show  the  existence  of  a  large  nucleus  situ- 
ated in  the  centre  of  the  cell  and  constituting  its  great 
hulk.  The  cell-wall  generally  is  not  stained,  but  when 
it  does  tinge,  a  delicate  line  of  unstained  material  can 
tiim-s  IK-  made  out  between  the  nucleus  and  the  cell- 
wall,  showing  the  existence  of  a  protoplasm. 

The-  anil  in  dyes,  which  possess  a  great  penetrating 
power,  color  the  organisms  so  intensely  as  to  preclude 
the  diffcn-miation  of  the  cellular  constituents.  Under 

30 


BACTERIA:  31 

these   conditions   the  bacteria  appear  as  solidly-colored 
spheres,  rods,  or  spirals,  as  the  case  may  be. 

The  cell-walls  of  some  of  the  bacteria  seem  at  times  to 
undergo  a  peculiar  gelatinous  change  or  to  allow  the  ex- 
'udation  of  gelatinous  material  from  the  protoplasm,  so 
that  the  individuals  appear  surrounded  by  a  distinct  halo 
or  capsule.  This  is  not  only  a  peculiarity  of  certain  indi- 
viduals, but  one  which  only  takes  place  when  they  develop 
under  certain  conditions;  thus,  Friedlander  points  out 
that  the  capsule  of  his  pneumonia  bacillus,  when  it  was 
found  in  the  lung  or  in  th*  "prune-juice"  sputum,  was 
very  distinct,  while  it  could  not  be  demonstrated  at  all 
when  the  organisms  grew  in  gelatin. 

From  the  cell-walls  of  many  bacteria  numerous  deli- 
cate straight  or  wavy  filaments  project.  These  are  called 
cilia  or  flagella,  and  seem  to  be  organs  of  locomotion. 
Sometimes  they  are  only  observed  projecting  from  the 
ends  or  from  one  end;  sometimes  they  are  so  numerous 
and  so  regular  in  their  distribution  as  to  give  the  organ- 
isms a  woolly  appearance. 

Many  of  the  bacteria  which  are  thus  supplied  with 
flagella  are  actively  motile  and  swim  about  like  mi- 
croscopic serpents.  In  all  probability  the  locomotory 
powers  of  the  bacteria  are  not  entirely  dependent  upon 
the  presence  of  the  flagella,  but  may  sometimes  be  due 
to  contractility  of  the  protoplasm  within  an  elastic  cell- 
wall.  The  micro-organisms  most  plentifully  supplied 
with  them  are  those  of  the  rod  and  spiral  shape.  Only 
one  of  the  spherical  forms,  Micrococcus  agilis  of  Ali- 
Cohen,  has  been  shown  to  have  flagella.  This  and  one 
other  species  are  probably  the  only  motile  cocci.  Ob- 
serving that  the  organisms  known  to  be  most  active  are 
those  best  supplied  with  flagella,  it  is  reasonable  to  con- 
clude that  the  motility  is  dependent  upon  the  flagella. 

The  presence  of  flagella,  however,  does  not  necessarily 
imply  motility,  for  some  of  the  bacilli  amply  provided 
with  these  appendages  are  not  motile.  The  flagella  may 
not  only  serve  as  organs  of  locomotion,  and  be  of  use  to 


32  PATHOGENIC  BACTERIA. 

the  organism  by  conveying  it  from  an  area  where  the 
nutrition  is  less  to  one  where  it  is  greater,  but,  .as  Wood- 
head  points  out,  may,  in  the  non-motile  species,  serve  to 
stimulate  the  passage  of  currents  of  nutrient  material 
past  the  organism,  so  as  to  increase  the  food-supply. 
The  flagellate  bacteria  have  a  greater  number  of  repre- 
sentatives among  those  whose  lives  are  "spent  in  water 
and  in  fermenting  and  decaying  materials  than  among 
those  inhabiting  the  bodies  of  animals.  This  is  an 
additional  fact  in  favor  of  the  view  that  locomotion  and 
flagella  are  provisions  favorable  to  the  maintenance  of 
the  species  by  keeping  the  individuals  supplied  with 
food. 

It  may  be  added  that  such  parasitic  disease-producing 
bacteria  as  do  not  habitually  gain  access  to  the  tissues, 
but  inhabit  the  intestine,  as  the  bacillus  of  typhoid  fever 
and  the  spirillum  of  cholera,  are  actively  motile,  like 
the  saprophytes,  while  those  habitually  entering  the  tis- 
sues and  multiplying  there  are  motionless  and  without 
flagella.  Of  course  this  example  is  open  to  criticism, 
because  the  spirillum  of  relapsing  fever,  which  has  never 
been  found  elsewhere  than  in  the  blood  and  spleen  of 
affected  animals,  is  actively  motile. 

One  of  the  linear  organisms,  known  as  the  Bacillus 
megatherium,  has  a  distinct  but  limited  ameboid  move- 
ment. 

The  commonly  observed  dancing  movement  of  the 
spherical  forms  seems  to  be  the  well-known  Brownian 
movement,  which  is  simply  a  physical  phenomenon.  It 
is  sometimes  difficult  to  determine  whether  an  organism 
is  really  motile  or  whether  it  is  only  vibrating.  In  the 
latter  case  it  does  not  change  its  relative  position  to 
surrounding  objects. 

The  bacteria  are  so  minute  that  a  special  unit  of  meas- 
urement has  been  adopted  by  bacteriologists  for  their 
estimation.  This  is  the  micro-millimeter  (//),  or  one- 
thousandth  part  of  a  millimeter,  and  about  equivalent 
to  the  one-twenty-five-thousandth  of  an  inch. 


BACTERIA.  33 

As  a  rule,  the  spherical  organisms  are  the  smallest  and 
the  spiral  organisms  the  longest,  except  the  chains  of 
bacilli  called  leptothri.r.  Their  measurements  vary  from 
0.15  /J.  (micrococcus  of  progressive  abscess-formation  in 
rabbits)  to  2.8  p.  (Diplococcus  albicans  ampins)  for  cocci, 
and  from  i  X  0.2  fJ.  (bacillus  of  mouse-septicemia)  to 
5  X  1.5  fj.  (anthrax  bacillus)  for  bacilli.  Some  of  the 
spirilla  are  very  long,  that  of  relapsing  fever  measuring 
40  fj.  at  times. 

This  estimation  of  size  almost  prepares  one  for  the 
estimation  of  weight  given  by  Nageli,  who  found  that 
an  average  bacterium  under  ordinary  conditions  weighed 
f  a  milligram. 


The  bacteria  multiply  in  two  ways  :  by  direct  division 
(fission)  and  by  the  development  of  spores,  seeds,  or  eggs 
(sporulation).  The  more  common  mode  is  by  binary 
division.  The  bacterium  which  is  about  to  divide  ap- 
pears a  little  larger  than  normal,  and,  if  a  spherical 
organism,  more  or  less  ovoid.  No  karyokinetic  changes 
have  been  observed  in  the  nuclei,  though  they  may  occur. 
When  the  conditions  of  nutrition  are  good,  the  process  of 
fission  progresses  with  astonishing  rapidity.  Buchner 
and  others  have  determined  the  length  of  a  generation 
to  be  from  fifteen  to  forty  minutes. 

The  results  of  binary  division,  if  rapidly  repeated,  are 
almost  appalling.  "  Cohn  calculated  that  a  single  germ 
could  produce  by  simple  fission  two  of  its  kind  in  an 
hour  ;  in  the  second  hour  these  would  be  multiplied  to 
four  ;  and  in  three  days  they  would,  if  their  surroundings 
were  ideally  favorable,  form  a  mass  which  can  scarcely  be 
reckoned  in  numbers,  or,  if  reckoned,  could  scarcely  be 
imagined  —  four  thousand  seven  hundred  and  seventy-two 
billions.  If  we  reduce  this  number  to  weight,  we  find 
that  the  mass  arising  from  this  single  germ  would  in 
three  days  weigh  no  less  than  seventy-five  hundred 
tons."  "Fortunately  for  us,"  says  Woodhead,  "they 
can  seldom  get  food  enough  to  carry  on  this  appalling 
rate  of  development,  and  a  great  number  die  both  for 


'34  /'.•/  THOGENIC  BACTERIA. 

want  of  food  and  because  of  the  presence  of  other  con- 
ditions unfavorable  to  their  existence." 

When  the  conditions  for  rapid  multiplication  are  no 
longer  good,  the  organism  assumes  a  protective  attitude 
and  develops  in  its  interior  small  oval  eggs,  seeds,  or,  as 
tlu-y  are  more  correctly  called,  spores  (Fig.  i).  Such 


o  o 


1. 1,..  i. Diagram  illustrating  sporulation  :  «,  bacillus  enclosing  a  small  oval 

spore;  b,  drumstick  bacillus,  with  the  spore  at  the  end;  c,  clostridium;  dy  free 
spores ;  e  and  /  bacilli  escaping  from  spores. 

spores  developed  within  the  bacteria  are  called  endospores. 
When  the  formation  of  such  a  spore  is  about  to  com- 
mence, a  small  bright  point  appears  in  the  protoplasm, 
and  increases  in  size  until  its  diameter  is  nearly  or  quite 
as  great  as  that  of  the  bacterium.  As  it  nears  perfection 
a  dark,  highly-refracting  capsule  is  formed  about  it.  As 
soon  as  the  spore  arrives  at  perfection  the  bacterium 
seems  to  die,  as  if  its  vitality  were  exhausted  in  the 
development  of  the  permanent  form. 

Endospores  are  generally  formed  in  the  elongate  bac- 
teria— bacillus  and  spirillum — but  Zopf  has  described 
similar  bodies  as  occurring  in  micrococci.  Escherich 
also  claims  to  have  found  undoubted  spores  in  a  form 
of  sarcina. 

The  spores  found  in  the  bacilli  are  either  round  or 
oval.  As  a  rule,  each  bacillus  produces  a  single  spore, 
which  is  situated  either  at  its  centre  or  at  its  end.  When, 
as  sometiiiR-s  happens,  the  diameter  of  the  spore  is  greater 
than  the  diameter  of  the  bacillus,  it  causes  a  bulging  of 
tin-  organism,  with  a  peculiar  appearance  described  as 
(lostfidiion.  When  the  distending-  spore  is  in  the  centre 
of  the  bacillus,  it  produces  a  barrel-shaped  organism; 
whin  situated  at  the  end,  a  "Trommelschlager,"  or  drum- 
stick->li  aped  one.  As  the  degeneration  of  the  protoplasm 
of  the  bacillus  sets  the  spore  free,  it  appears  as  a  clear,. 


BACTERIA.  35 

highly-refracting  sphere  or  ovoid  situated  in  a  little  col- 
lection of  granular  matter. 

Spores  differ  from  the  bacteria  in  that  their  capsules 
seem  to  prevent  evaporation  and  to  enable  them  to  with- 
stand drying  and  the  application  of  a  considerable  amount 
of  heat.  Ordinarily,  bacteria  are  unable  to  resist  a  tem- 
perature above  60°  C.  for  any  considerable  length  of 
time,  only  a  few  resistant  forms  tolerating  a  temperature 
of  70°  C.  The  spores,  however,  are  uninjured  by  such 
temperatures,  and  can  even  successfully  resist  that  of 
boiling  water  (100°  C.)  for  a  short  time.  The  extreme 
desiccation  caused  by  a  protracted  exposure  to  a  tem- 
perature of  150°  C.  will,  however,  destroy  them.  Not  only 
can  the  spores  resist  a  considerable  degree  of  heat,  but 
they  are  also  unaffected  by  cold  of  almost  any  intensity. 

While  the  cell-wall  of  the  bacterium  is  easily  pene- 
trated by  solutions  of  the  anilin  dyes,  it  is  a  matter  of 
much  difficulty  to  accomplish  the  staining  of  spores,  so 
that  we  see  they  are  probably  more  resistant  to  the 
action  of  chemical  agents  than  the  bacteria  themselves. 

When  a  spore  is  accidentally  dropped  into  some  nu- 
trient medium  a  change  is  shortly  observed.  The  proto- 
plasm, which  has  been  clear,  becomes  somewhat  granu- 
lar, the  capsule  a  little  less  distinct;  the  body  increases 
slightly  in  size,  and  in  the  course  of  time  splits  open  to 
allow  the  escape  of  the  young  organism.  The  direction 
in  which  the  escape  of  the  young  bacillus  takes  place  is 
of  interest,  as  varying  in  the  different  species.  The 
Bacillus  subtilis  escapes  from  the  end  of  the  spore,  where 
a  longitudinal  fissure  occurs;  the  bacillus  of  anthrax 
escapes  from  the  side,  sometimes  leaving  the  capsule  of 
the  spore  in  the  shape  of  two  small  cups. 

As  soon  as  the  young  bacillus  escapes  it  begins  to  in- 
crease in  size,  develops  around  its  soft  protoplasm  a  cha- 
racteristic capsule,  and,  having  once  established  itself, 
presently  begins  the  propagation  of  its  species  by  fission. 

In  addition  to  the  endospores,  of  which  we  have  just 
been  speaking,  there  are  arthrospores.  The  formation 


36  PATHOGENIC  BACTERIA. 

of  these  is  much  less  clear.  It  seems  to  be  an  effort  to 
convert  the  entire  microbe  into  a  permanent  form.  This 
process  is  observed  particularly  in  the  micrococci,  where 
the  substance  of  a  cell  is  said  to  break  up  into  segments, 
each  of  which  becomes  a  resisting  body  fruitful  in  prop- 
agating its  species.  Of  the  arthrospores  little  has,  so 
far.  been  learned.  It  is  not  improbable  that  among  the 
micrococci,  and  also  among  some  of  the  smaller  bacilli 
in  whom  no  spores  have  been  observed,  the  maintenance 
of  the  species  when  conditions  of  life  become  unfavor- 
able is  due  to  the  assumption  of  a  permanent  form  by 
some  of  the  individuals,  without  the  formation  of  any 
spore-like  bodies.  This  is  at  present  largely  a  matter  of 
conjecture,  but  the  indications  pointing  in  that  direction 
are  numerous. 

It  is  believed  by  Frankel  and  others  that  sporulation 
in  the  bacteria  is  not  a  sign  of  the  exhaustion  of  nutri- 
tion, but  a  sign  of  the  vital  perfection  of  the  organism. 
These  observers  regard  spore-formation  as  analogous  to 
the  flowering  of  higher  plants,  which  takes  place  only 
when  the  conditions  and  development  are  best. 

Morphology.  —  The  morphology  of  the  bacteria  is  quite 
varied.  Three  principal  forms,  however,  exist,  from  which 
the  others  seem  to  be  but  variations. 

The  most  simple  appear  as  minute  spheres,  and  from 


0  O 

1MB    ill.istr.ili..-    the    ......plH.lnjjy  of  ,1K.    OK.ci.   (/<  0)Cais   <)r 

IKOCCUS;  *tdip],,,-  ,   ,,rt.pt«cocci;  et  /  tetragenococci  or  meris- 

mopedia;  g,  k,  modes  of  division  of  cocci;  /.  Htrcina  ;  /.  COCCUS  with  flagdla- 
*,  uUphylococci. 

tlu-ir  fancied  ivsanblauce  to  little  berries  are  called  cocci 
<>i  m  (Fig.  2,  a).     When  the  bacteria  of  this  form 


BACTERIA.  37 

multiply  by  fission  the  resulting  two  organisms  not 
infrequently  remain  attached  to  each  other,  producing 
what  is  called  a  diplococcus  (Fig.  2,  b).  The  diplococci 
sometimes  consist  of  two  perfect  spheres,  but  more  often 
show  a  flattening  of  the  contiguous  surfaces,  which  are 
not  in  absolute  apposition  (Fig.  2,  g).  In  a  few  cases,  as 
the  gonococcus,  the  approximated  surfaces  are  slightly 
concave,  causing  the  organism  to  somewhat  resemble  the 
German  biscuit  called  a  "semmel,"  hence  biscuit-  or 
semmel-cocci  (Fig.  2,  //).  Frequently  a  second  binary  di- 
vision occurs,  causing  four  individuals  to  remain  closely 
approximated,  without  disturbing  the  arrangement  of  the 
first  two.  When  division  of  this  kind  produces  a  distinct 
tetrad,  the  organism  is  described  as  a  tetragenococcus, 
while  to  the  entire  class  of  cocci  dividing  so  as  to  pro- 
duce fours,  eights,  twelves,  etc.  on  the  same  plane  the 
name  merismopedia  is  given  (Fig.  2,  e  and  f). 

If,  as  sometimes  happens,  the  divisions  take  place  in 
three  directions,  so  as  to  produce  cubical  masses  or  "pack- 
ages" of  cocci,  the  resulting  aggregation  is  described  as 
a  sarcina  (Fig.  2,  t).  This  form  slightly  resembles  a  dice 
or  a  bale  of  cotton  in  miniature. 

If  the  divisions  always  take  place  in  the  same  direc- 
tion, so  as  to  produce  a  chain  or  string  of  beads,  the 
organism  is  described  as  streptococcus  (Fig.  2,  d\  When 
there  are  diplococci  joined  in  this  manner  a  strepto-diplo- 
coccus  is  of  course  formed. 

More  common  than  any  of  the  forms  already  described 
is  one  in  which,  without  any  definite  arrangement,  the 
cocci  occur  in  irregular  groups  having  a  fancied  resem- 
blance to  bunches  of  grapes.  These  are  called  staphylo- 
cocci,  and,  as  it  is  very  unusual  to  find  cocci  habitually 
occurring  isolated,  most  cocci  not  classified  under  one  of 
the  above  heads  are  called  staphylococci. 

When  cocci  are  associated  in  globular  or  lobulated 
clusters  encased  in  a  resisting  glutinous,  homogeneous 
mass,  the  name  ascococcus  has  been  used  in  describing 
them.  A  modified  form  of  this,  in  which  the  cocci  are 


38  PATHOGENIC  BACTERIA. 

in  chains  or  solitary  and  are  surrounded  by  an  encase- 
ment almost  cartilaginous  in  consistence,  has  been  called 
leuconostoc. 

Certain  bacteria,  constituting  a  better-known  if  not 
more  important  group,  are  not  spherical,  but  elongate 
or  "  rod-shaped,"  and  bear  the  name  bacillus  (Fig.  3). 


FIG.  3. — Diagram  illustrating  the  morphology  of  the  bacilli :  a,  b,  c,  various 
forms  of  bacilli ;  d,  e,  bacilli  with  flagella ;  /,  chain  of  bacilli,  individuals  dis- 
tinct; gt  chain  of  bacilli,  individuals  not  separated. 

I  would  remark  that  the  absence  of  a  standard  by 
which  to  separate  the  cocci  from  the  bacilli  is  the  cause 
of  much  confusion.  In  the  judgment  of  the  author,  it 
would  be  well  to  place  all  individuals  having  one  diam- 
eter greater  than  the  other  among  the  bacilli.  This 
would  prevent  the  error  of  describing  one  species  as 
44 oval  cocci n  and  another  as  "nearly  round  bacilli," 
and  by  giving  a  definite  standard  would  greatly  aid  in 
the  formation  of  a  differential  table. 

The  bacilli  present  a  considerable  variety  of  forms. 
Some  are  quite  short,  with  rounded  ends,  so  as  to  ap- 
pear elliptical ;  some  are  long  and  delicate.  Some  have 
rounded  ends,  as  subtilis  ;  others  have  square  ends,  as 
anthrax.  Some  are  enormously  large,  some  exceedingly 
small.  Some  are  always  isolated,  never  forming  threads 
or  chains  ;  others  nearly  always  occur  in  these  forms. 

The  bacilli  always  divide  by  transverse  fission,  so  that 
the  only  peculiarity  of  arrangement  is  the  formation  of 
threads  or  chains. 

In  the  older  writings  the  short,  stout  bacilli  were  all 
described  under  the  generic  term  bacterium.  This  genus, 
like  some  of  the  species  it  comprehended,  has  now  passed 


BACTERIA.  39 

out  of  use.  Some  of  the  flexile  bacilli,  whose  movements 
are  sinuous,  much  resembling  the  swimming  of  a  snake 
or  an  eel,  were  described  as  vibrio,  but  this  name  also  has 
passed  into  disuse. 

The  long  filaments  formed  by  the  division  of  bacilli 
without  their  distinct  separation  are  sometimes  called 
leptothrix,  and  when  these  long  threads  form  distinct 
masses  surrounded  by  a  jelly-like  material,  the  name 
myconostoc  is  sometimes  applied  to  them. 

Certain  forms  much  resembling  bacilli  in  their  isolated 
state,  characterized  by  the  formation  of  long  filaments 
with  a  peculiar  grouping  which  gives  the  appearance 
of  a  false  branching,  are  described  as  cladothrix ;  others 
in  which  true  branchings  are  seen,  as  streptothrix.  One 
other  bacillus-like  form,  consisting  of  long,  thick,  not 
distinctly  segmented,  straight  threads,  is  called  beggiatoa. 
The  only  important  difference  between  it  and  leptothrix 
is  that  its  filaments  are  thick  and  coarse,  while  those  of 
leptothrix  are  very  delicate. 

Some  of  the  elongate  bacteria  have  a  remarkably 
twisted  form  and  bear  some  resemblance  to  a  cork- 
screw. These  are  called  spirilla  (Fig.  4).  A  subdivision 


FlG.  4. — Diagram  illustrating  the  morphology  of  the  spirilla:  a,  b,  c,  spirilla; 
d,  e,  spirochseta. 

of  them,  whose  individuals  are  not  only  twisted  but  are 
also  very  flexible,  is  called  spiroch&ta.  Though  not 
formerly  differentiated  from  vibrio,  these  forms  are  quite 
distinct. 

A  spiral  organism  of  a  ribbon  shape  is  called  spiro- 


40  PATHOGENIC  BACTERIA. 

HHHHIS,  while  a  similar  organism  of  spindle  shape  is 
called  a  spirulhni.  One  species  of  spiral  bacteria  in 
whose  protoplasm  sulphur-grounds  have  been  detected 
has  been  called  ophidiomonas. 

Some  of  the  spirilla  are  exceedingly  long  and  deli- 
cate, as  the  spirochaeta  of  relapsing  fever ;  others  which 
are  stouter,  like  the  spirillum  of  cholera,  habitually  occur 
in  such  short  individuals  as  to  be  easily  mistaken  for 
slightly-bent  bacilli. 

Classification. — Leeuwenhoek,  when  he  first  saw  the 
bacteria — and  his  successors  even  to  so  recent  a  date  as 
to  include  Ehrenberg  and  Dujardin — did  not  doubt  that 
they  belonged  to  the  infusoria. 

It  was  not  until  biologists  had  concluded  that  organ- 
isms which  take  into  their  bodies  particles  of  solid  or 
semi-solid  material,  digest  that  which  is  useful,  and 
extrude  the  remainder,  are  animals,  and  that  those  which 
live  purely  by  osmosis  and  exosmosis  are  vegetables,  that 
the  bacteria,  which  we  have  seen  provided  with  a  resist- 
ant cell-wall,  allowing  of  no  possibility  of  nutrition 
except  by  osmosis  and  exosmosis,  could  be  finally  and 
correctly  classed  among  the  members  of  the  vegetable 
kingdom. 

The  extremely  simple  organization  of  bacteria  naturally 
places  them  among  the  lowest  members  of  the  vegetable 
kingdom,  in  that  class  of  the  Cryptogamia  known  as 
Thallophytae,  comprising  the  algae,  lichens,  and  fungi. 

The  algae  are  mostly  water-plants,  containing  chloro- 
phyl  and  obtaining  their  nourishment  from  inorganic 
substances. 

The  lichens  are  plants,  some  of  which  contain  chloro- 
phyl.  They  live  upon  inorganic  matter,  which  they 
generally  absorb  from  the  air.  According  to  the  new 
view  of  the  subject,  some,  if  not  all,  of  these  plants  are 
regarded  as  fungi  growing  parasitically  upon  algae. 

The  fun-i,  the  lowest  group  of  all,  are  minute  or  large 
plants,  mostly  devoid  of  chlorophyl,  living  upon  organic 
matter,  which  they  obtain  as  saprophytes  from  decom- 


BACTERIA.  41 

posing   animal   and   vegetable   matters,   or   as   parasites 
upon  the  tissues  or  juices  of  living  animals  or  plants. 
This  lowest  family,  the  fungi,  are  divisible  into  the — 

Hyphomycetes  or  Mucorini,  or  moulds; 
Saccharomycetes,  or  yeasts;  and 
Schizomycetes,  or  bacteria. 

Cohn  divided  the  bacteria,  according  to  their  mor- 
phology, into — 

Sphero-bacteria,  or  cocci ; 
Micro-bacteria — short  rods  ; 
Desmo-bacteria — bacilli ; 
Spiro-bacteria — spirilla-. 

Davaine  suggested  a  classification  based  upon  motility, 
making  four  classes — Bacterium,  Vibrio,  Bacteridium, 
and  Spirillum,  neglecting  to  provide  for  the  cocci. 

Zopf  arranged  them,  according  to  his  theory  of 
pleomorphism,  into  the  CocCACE^E,  comprising  those 
known  only  in  the  coccus  form,  and  comprehending 
the  streptococci,  merismopedia,  sarcina,  micrococcus,  and 
ascococcus ;  the  BACTERIACE^,  comprehending  the  genera 
bacterium,  spirillum,  vibrio,  leuconostoc,  bacillus,  and 
clostridium  (chiefly  coccus,  rod,  and  thread  forms  ;  the 
former  may  be  absent ;  in  the  latter  there  is  no  distinction 
between  base  and  apex  ;  threads  straight  or  screw-like)  ; 
and  the  LEPTOTHRICHE.^,  comprehending  crenothrix, 
beggiatoa,  phragmidiothrix,  and  leptothrix  (coccus,  rod, 
and  thread  forms  ;  the  latter  show  a  distinction  between 
base  and  apex  ;  threads  straight  or  screw-like  ;  spore- 
formation  not  demonstrated). 

This  classification  is,  however,  based  upon  what  is 
probably  an  erroneous  principle,  the  pleomorphism  of 
the  bacteria. 

Van  Tieghem,  DeBary,  and  Hiippe  formed  classifica- 
tions the  main  feature  of  which  was  the  formation  of 
endospores  or  arthrospores,  but,  as  the  sporulation  of 
many  species  is  as  yet  unknown,  they  cannot  be  properly 
placed  in  it. 


42  PA  THOGENIC  BA  CTERIA. 

It  has  even  been  suggested  to  classify  the  bacteria  by 
the  size  and  number  of  their  flagella,  of  which  so  little 
is  known. 

The  most  convenient  classification,  though  it  cannot 
be  purely  scientific,  seems  to  be  the  morphological  one 
given  by  Colin.  Baumgarten,  recognizing  the  relative 
pleomorphism  of  certain  of  the  species,  has  modified  it 
as  follows,  and  thus  made  it  answer  all  the  needs  of  the 
pathologist  at  least: 

I.  Cocci,  \ 

II.  Bacilli,  >  species  relatively  monomorphous. 

III.  Spirilla,  ) 

IV.  Spirulina,  \ 

Y.  Leptothrix,  [•  species  relatively  pleomorphous. 
VI.  Cladothrix, ) 

The  members  of  the  first  group,  the  cocci,  bacilli,  and 
spirilla,  are  practically  the  only  ones  which  are  of  patho- 
logical significance. 


CHAPTER   II. 
BIOLOGY   OF  BACTERIA. 

THE  distribution  of  bacteria  is  wellnigh  universal. 
They  and  their  spores  float  in  the  atmosphere  we  breathe, 
swim  in  the  water  we  drink,  grow  upon  the  food  we  eat, 
and  luxuriate  in  the  soil  beneath  our  feet.  Nor  is  this 
all,  for,  entering  the  palpebral  fissures,  they  develop  upon 
the  conjunctiva  ;  entering  the  nares,  they  establish  them- 
selves in  the  nose  ;  the  mouth  is  always  replete  with 
them  ;  and,  as  many  are  swallowed,  the  digestive  appa- 
ratus always  contains  them.  The  surface  of  the  body 
never  escapes  their  establishment,  and  so  deeply  are 
some  individuals  situated  beneath  the  epithelial  cells 
that  the  most  careful  washing  and  scrubbing  and  the  use 
of  the  most  powerful  germicides  are  required  to  rid  the 
surgeon's  hands  of  what  may  prove  to  be  dangerous 
hindrances  to  the  healing  of  wounds.  The  ear  is  not 
without  its  microscopic  flora  ;  special  varieties  live  be- 
neath the  finger-nails,  and  especially  the  toe-nails,  in 
the  vagina,  and  beneath  the  prepuce. 

While  so  general,  however,  they  are  not  ubiquitous. 
Tyndall  succeeded  in  proving  that  the  atmosphere  of 
high  Alpine  altitudes  was  free  from  them,  and  likewise 
that  the  glacier  ice  contained  none.  Wherever  man,  ani- 
mals, or  even  plants,  live,  die,  and  decompose,  bacteria 
are  sure  to  be  present. 

Notwithstanding  their  extreme  familiarity  with  the 
animal  body,  there  are  certain  parts  of  it  into  which 
bacteria  do  not  enter,  or,  entering,  remain  vital  for  a 
very  short  time,  for  the  body-juices  and  tissiies  of  normal 
animals  are  free  from  them,  and  their  occurrence  there 
may  almost  always  be  accepted  as  a  sign  of  disease. 

The  presence  of  bacteria  in   the  air  is  generally  de- 

43 


44  PA  THOGENIC  BACTERIA . 

pendant  upon  their  previous  existence  in  the  soil,  its  pul- 
verization, and  its  distribution  by  currents  of  the  atmo- 
sphere. Koch  has  shown  that  the  upper  stratum  of  the 
soil  is  exceedingly  rich  in  bacteria,  but  that  their  num- 
bers decrease  as  the  soil  is  penetrated,  until  below  a 
depth  of  one  meter  there  are  very  few.  Remembering 
that  bacteria  can  live  only  upon  organic  matter,  this  is 
readily  understandable.  Most  of  the  organic  matter  is 
upon  the  surface  of  the  soil.  Where,  as  in  the  case  of 
porous  soil  or  the  presence  of  cesspools  and  dung-heaps, 
the  decomposing  materials  are  allowed  to  penetrate  to  a 
considerable  depth,  the  bacteria  may  occur  much  farther 
from  the  surface,  yet  they  are  rarely  found  at  any  great 
depth,  because  the  majority  of  the  known  species  require 
oxygen. 

The  water  of  stagnant  pools  always  teems  with  bacte- 
ria, but  that  of  deep  wells  rarely  contains  many  unless 
it  is  polluted  from  the  surface  of  the  earth. 

Being  generally  present  in  the  soil,  which  the  feet  of 
men  and  animals  grind  to  powder,  the  bacteria,  together 
with  the  pulverized  earth,  are  blown  from  place  to  place 
into  every  nook  and  cranny,  until  it  is  impossible  to  es- 
cape them.  It  has  been  suggested  by  Soyka  that  the 
currents  of  air  passing  over  the  surface  of  liquids  might 
take  up  bacteria,  but,  although  he  seemed  to  show  it  ex- 
perimentally, it  is  not  generally  believed.  Where  bac- 
teria are  growing  in  colonies  they  seem  to  remain  un- 
disturbed bv  currents  of  air  unless  the  surface  becomes 
roughened  or  broken. 

M«>st  of  the  bacteria  which  are  carried  about  by  the  air 
are  what  are  called  saprophytes,  and  are  perfectly  harm- 
less to  the  human  being;  but  not  all  belong  to  this  class, 
nor  will  they  do  so  while  tuberculous  patients  are  al- 
lowed to  expectorate  upon  the  sidewalks,  and  typhoid 
patient,'  w.ish  to  dry  upon  the  clothes-line,  and  their 
dejecta  to  be  spread  upon  the  ground. 

The  growth  of  bacteria  is  profoundly  influenced  by 
environment,  so  that  a  consideration  of  the  conditions 


BIOLOGY  OF  BACTERIA.  45 

favorable  or  detrimental  to  their  existence  becomes  a 
necessity. 

Conditions  influencing  the  Growth  of  Bacteria. — 
(a)  Oxygen. — The  majority  of  bacteria  grow  best  when 
exposed  to  the  air.  Some  develop  better  when  the  air  is 
withheld;  some  will  not  grow  at  all  where  the  least 
amount  of  oxygen  is  present.  Because  of  these  pecu- 
liarities bacteria  are  divisible  into  the 

Aerobic  bacteria,  those  growing  in  oxygen. 

Anaerobic  bacteria,  those  not  growing  in  the  presence 
of  oxygen. 

As,  however,  some  of  the  aerobic  forms  will  grow 
almost  as  well  without  as  with  oxygen,  the  term  optional 
(facultative)  anaerobics  has  been  applied  to  the  special 
class  made  to  include  them. 

As  examples  of  strictly  aerobic  bacteria  the  Bacillus 
subtilis  and  the  Bacillus  aerophilus  may  be  given.  These 
forms  will  not  grow  if  oxygen  is  denied  them.  The 
staphylococci  of  suppuration  and  the  bacilli  of  typhoid 
fever,  pneumonia,  and  anthrax,  as  well  as  the  spirillum 
of  cholera,  will  grow  almost  equally  well  with  or  with- 
out oxygen,  and  hence  belong  to  the  optional  anaerobics. 
The  bacillus  of  tetanus  and  of  malignant  edema,  and  the 
non-pathogenic  forms,  the  Bacillus  butyricus,  Bacillus 
muscoides,  and  Bacillus  polypiformis,  will  not  develop 
at  all  where  any  oxygen  is  present,  and  hence  are 
strictly  anaerobic. 

(6}  Nutriment.—  The  bacteria  do  not  seem  able  to  derive 
their  nourishment  from  purely  inorganic  matter.  Pros- 
kauer  and  Beck,  however,  have  succeeded  in  growing  the 
tubercle  bacillus  in  a  mixture  containing  ammonium 
carbonate  0.35  per  cent,  potassium  phosphate  0.15  per 
cent.,  magnesium  sulphate  0.25  per  cent,  glycerin  1.5 
per  cent.  They  grow  best  where  diffusible  albumins  are 
present.  The  ammonium  salts  are  rather  less  fitted  to 
support  them  than  their  organic  compounds.  The  in- 
dividual bacterium  varies  very  widely  in  the  nutriment 
-which  it  requires.  Some  of  the  water-microbes  can  live 


46  PATHOGENIC  BACTERIA. 

in  distilled  water  to  which  the  smallest  amount  of  organic 
matter  has  been  added;  others  require  so  concentrated  a 
medium  that  only  blood-serum  can  be  used  for  their 
cultivation.  Sometimes  a  species  with  a  preference  for  a 
particular  culture-medium  can  gradually  be  accustomed 
to  another,  though  immediate  transplantation  causes  the 
death  of  the  transplanted  organism.  Sometimes  the  ad- 
dition of  such  substances  as  glucose  and  glycerin  has  a 
peculiarly  favorable  influence  upon  bacteria,  causing,  for 
example,  the  tubercle  bacillus  to  grow  upon  agar-agar. 

(r)  Moisture. — A  certain  amount  of  water  is  always 
necessary  for  the  growth  of  bacteria.  The  amount  can 
be  exceedingly  small,  however,  so  that  the  Bacillus  pro- 
digiosus  is  able  to  develop  successfully  upon  crackers  and 
dried  bread.  Materials  used  as  culture-media  should  not 
be  too  concentrated;  at  least  80  per  cent,  of  water  should 
be  present.  Most  bacteria  grow  best  in  liquid  media; 
that  is,  they  form  the  longest  threads,  and  diffuse  them- 
selves throughout  the  liquid  so  as  to  be  present  in  far 
greater  numbers  than  when  on  solid  media. 

The  statement  that  certain  forms  of  bacteria  can  flour- 
ish in  clean  distilled  water  seems  to  be  untrue.  When 
transferred  to  such  a  medium  the  organisms  soon  die  and 
undergo  a  granular  degeneration  of  their  substance.  If, 
however,  in  their  introduction  a  good-sized  drop  of  cul- 
ture-material is  carried  with  them,  the  distilled  water 
ceases  to  be  such,  and  becomes  a  dilute  bouillon  fitted  to 
support  life  for  a  time. 

(//)  Reaction. — Should  the  pabulum  supplied  to  bacte- 
ria contain  an  excess  of  either  alkali  or  acid,  the  growth 
of  the  organisms  is  inhibited.  Most  true  bacteria  grow 
best  in  a  neutral  or  feebly  alkaline  medium.  There  are 
exceptions  to  this  rule,  for  the  Bacillus  butyricus  and  the 
Sarcina  ventriculi  can  grow  well  in  strong  acids,  and  the 
urea  ran  tolerate  excessive  alkalinity.  Acid 
media  are  excellent  for  the  cultivation  of  moulds. 

(e)  Light. — Most  species  of  bacteria  are  not  influenced 
in  their  growth  by  the  presence  or  absence  of  light.  The 


BIOLOGY  OF  BACTERIA.  47 

direct  rays  of  the  sun,  and  to  a  less  degree  the  intense 
rays  of  the  electric  arc-light,  retard  and  in  numerous  in- 
stances kill  bacteria.  Some  colors  are  distinctly  inhibi- 
tory to  their  growth,  blue  being  especially  prejudicial. 
Some  of  the  chromogenic  forms  will  only  produce  their 
colors  when  exposed  to  the  ordinary  light  of  the  room. 
The  Bacillus  mycoides  roseus  will  not  produce  its  red 
pigment  except  in  the  absence  of  light.  The  pathogenic 
bacteria  have  their  virulence  gradually  attenuated  if 
grown  in  the  light. 

(f)  Electricity. — Very  little  is  known  about  the  action 
of  electric  currents  upon  bacteria.     Very  powerful  dis- 
charges of  electricity  through  culture-media  are  said  to 
kill  the  organisms,  to  change  the  reaction  of  the  culture, 
and    the  rapidly  reversed    currents  of  high  intensity  to 
destroy  the  pathogenesis  of  the  bacteria  and  change  their 
toxic  products  into   neutralizing  protective  (antitoxin?) 
bodies.     Much    attention    has  recently  been    devoted  to 
this  subject  by  Smirnow,  Arson val  and  Charin,   Bolton 
and  Pease,   Bonome  and  Viola,  and  others. 

(g)  Movement. — When  bacteria  are  growing  in  a  liquid 
medium    perfect   rest    seems   to   be   the    condition    best 
adapted  for  their  development.     A  slow-flowing   move- 
ment does  not  have  much  inhibitory  action,  but  violent 
agitation,  as  by  shaking  a  culture  in  a  machine,  greatly 
hinders  or  prevents  their  growth.     The  practical  appli- 
cation of  this  will  show   that  rapidly  flowing  streams, 
whose  currents  are  interrupted  by  falls  and  rapids,  will, 
other  things  being  equal,  furnish  a  better  drinking-water 
than  a  deep,  still-flowing  river. 

(//)  Association. — It  occasionally  happens  that  bacteria 
grow  better  when  associated  with  other  species,  or  have 
their  pathogenic  powers  augmented  when  grown  in  com- 
bination. Coley  found  the  streptococcus  toxin  more 
active  when  combined  with  Bacillus  prodigiosus. 

Occasionally  the  reverse  is  true,  and  Pawlowski  found 
that  mixtures  of  anthrax  and  bacillus  prodigiosus  were 
less  virulent  than  cultures  of  anthrax  alone. 


48  /'.  /  THOGENIC  BACTERIA. 

Rarely,  the  presence  of  one  species  of  microorganism 
entirely  eradicates  another  species.  Hankin  found  that 
tlu-  Micrococcns  Ghadialli  destroyed  the  typhoid  and  colon 
bacilli,  and  suggested  the  use  of  this  coccus  to  purify 
waters  polluted  with  typhoid.1 

(/)  7i  in/>t  rat itre. — The  question  of  temperature  is  of 
importance  from  its  bearing  upon  sterilization.  Accord- 
ing to  Frankel,  bacteria  will  scarcely  grow  at  all  below 
1 6°  and  above  40°  C. 

The  researches  of  Fliigge  show  that  the  Bacillus  sub- 
tilis  will  grow  very  slowly  at  6°  C.,  and  as  the  tempera- 
ture is  elevated  it  is  said  that  until  12.5°  C.  is  reached 
fission  does  not  occur  oftener  than  every  four  or  five 
hours.  When  25°  C.  is  reached  the  fission  occurs  every 
three-quarters  of  an  hour,  and  at  30°  C.  about  every  half 
hour. 

Most  bacteria  die  at  a  higher  temperature  than  60- 
75°  C.  The  spores  can  resist  boiling  water,  but  are 
killed  by  dry  heat  if  exposed  to  150°  C.  for  an  hour  or  to 
175°  C.  for  five  to  ten  minutes.  Freezing  kills  many,  but 
not  all  bacteria,  but  does  not  affect  the  spores  at  all. 

Most  bacteria  grow  best  at  the  ordinary  temperature  of 
a  comfortably  heated  room,  and  are  not  affected  by  its 
-ional  slight  changes.  Some,  chiefly  the  pathogenic 
forms,  are  not  cultivable  except  at  the  temperature  of 
the  animal  body  (37°  C.);  others,  like  the  tubercle  bacil- 
lus, grow  best  at  a  temperature  a  little  above  that  of  the 
body— 40°  C. 

Variations  in  the  amount  of  oxygen,  temperature,  moist- 
ure, etc.,  beyond  what  have  been  described,  are  prej- 
udicial to  the  growth  and  development  of  bacteria,  first 
inhibiting  their  growth,  thus  tending  toward  their  de- 
struction. In  the  practical  application  of  our  knowledge 
of  the  biology  of  the  bacteria  we  constantly  make  use  of 
such  precautions  as  removing  from  surgical  dressings, 
sponges,  etc.,  every  substance  that  can  possibly  afford 
nutriment  to  bacteria,  and  heating  such  materials,  as  well 

'•/.  Jour.,  Aug.  14,  1897,  p.  418. 


BIOLOGY  OF  BACTERIA.  49 

as  culture-media  arid  a  variety  of  other  substances,  to  a 
temperature  beyond  that  known  to  be  the  extreme  limit 
of  bacterial  endurance. 

The  presence  of  certain  substances — especially  some 
of  the  mineral  salts — in  an  otherwise  perfectly  suitable 
medium  will  prevent  the  development  of  bacteria,  and 
when  added  to  grown  cultures  of  bacteria  will  destroy 
them.  Carbolic  acid  and  bichlorid  of  mercury  are  the 
best  known  examples. 

It  is  interesting  to  mention  in  this  connection  the 
results  of  the  experiments  of  Trambusti,  who  found  it 
possible  to  produce  a  tolerance  to  a  certain  amount  of 
bichlorid  of  mercury  by  cultivating  Friedlander's  bacillus 
upon  culture-media,  containing  gradually  increasing 
amounts  of  the  salt,  until  from  1-15,000,  which  inhibited 
ordinary  cultures,  it  could  accommodate  itself  to  1-2000. 

(/)  x-Rays. — The  action  of  the  .r-rays  upon  bacteria 
has  been  investigated  by  Bonome  and  Gros  and  others. 
When  the  cultures  are  exposed  to  their  action  for  pro- 
longed periods,  their  vitality  and  virulence  seem  to  be 
slightly  diminished.  They  are  not  killed  by  the  .r-rays. 

Some  forms  of  the  bacteria  are  never  found  except  in 
the  tissues  of  diseased  animals.  Such  organisms  are 
called  parasites.  The  parasitic  group  really  is  divisible 
into  the  purely  parasitic  and  the  occasionally  parasitic 
bacteria.  Of  the  first  division  the  tubercle  bacillus  may 
be  used  as  an  illustration,  for,  so  far  as  is  known,  it  is 
never  found  in  other  places  than  the  bodies  and  dejecta 
of  diseased  animals.  The  cholera  spirillum  illustrates 
the  second  group,  for,  while  it  produces  the  disease 
known  as  Asiatic  cholera  when  admitted  to  the  digestive 
tract,  it  is  a  constant  inhabitant  of  certain  waters,  where 
it  multiplies  with  luxuriance. 

Bacteria  which  do  not  enter  the  animal  economy,  or  if 
accidentally  admitted  do  no  harm,  but  live  upon  decaying 
animal  and  vegetable  materials,  are  called  saprophytes. 

According  to  their  products  of  metabolism,  bacteria 
are  often  described  as — 

4 


5o  PA  THOGENIC  BA  CTERIA. 

Zymogenic,  or  bacteria  of  fermentation. 
Saprogenic,  or  bacteria  of  putrefaction. 
Chromogenic,  or  color  producers. 
Photogenic,  or  phosphorescent  bacteria. 
Aerogenic,  or  gas  producers. 
Pathogenic,  or  disease  producers. 

The  parasitic  organisms  alone  possess  much  interest  to 
the  physician,  but  as  in  their  growth  the  saprophytes  ex- 
hibit many  interesting  vital  manifestations,  it  is  not  well 
to  exclude  them  or  their  products  from  the  following 
consideration  of  the 

Results  of  Vita!  Activity  in  Bacteria.— i.  Fermenta- 
t,'t),lf — The  alcoholic  fermentation,  which  is  a  familiar  phe- 
nomenon to  the  layman  as  well  as  to  the  brewer  and  the 
chemist,  is  not  the  work  of  a  bacterium,  but  of  a  yeast- 
plant,  one  of  the  saccharomyces  fungi.  The  acetic-acid, 
lactic-acid,  and  butyric-acid  fermentations  are,  however, 
caused  by  bacilli.  A  considerable  number  of  bacilli  seem 
capable  of  converting  milk-sugar  into  lactic  acid,  some- 
times associating  this  with  coagulation  of  milk,  some- 
times not.  The  production  of  coagulation  in  milk  is  not 
always  associated  with  acid-production,  but  with  the  pro- 
duction of  a  curdling  ferment  similar  to  that  belonging 
to  the  gastric  juice.  There  seems  to  be  no  real  specific 
micro-organism  for  the  lactic-acid  fermentation,  although 
the  Bacillus  acidi  lactici  seems  to  be  the  most  powerful 
generator  of  the  acid.  There  may  also  be  several  bac- 
teria which  produce  the  acetic  fermentation,  though  it  is 
generally  attributed  to  a  special  common  form,  the  Myco- 
(k-nna  aceti  or  Bacillus  aceticus.  The  butyric  fermenta- 
tion is  generally  due  to  the  Bacillus  butyricus,  though  it 
also  may  be  caused  by  other  bacilli,  the  one  named  sim- 
ply being  the  most  common.  (For  an  exact  description 
of  the  chemistry  of  the  fermentations  reference  must  be 
in. uli-  to  u-xt-books  upon  that  subject,  as  their  considera- 
tion lu  iv  would  occupy  too  much  space.) 

2.   rut)<  fusion. — Tliis  process  is  in  many  respects  sim- 


BIOLOGY  OF  BACTERIA.  51 

ilar  to  the  preceding,  except  that  instead  of  occurring  in 
carbohydrates  it  takes  place  in  nitrogenous  bodies.  The 
first  step  seems  to  be  the  transformation  of  the  albumins 
into  peptones,  then  the  splitting  up  of  the  peptones  into 
a  large  number  of  gases,  acids,  bases,  and  salts.  In  the 
process  the  innocuous  albumins  are  frequently  changed  to 
toxalbumins,  and  sometimes  to  distinct  animal  alkaloids 
known  as  ptomaines.  Vaughan  and  Novy  declare  the 
term  "  animal  alkaloid  "  to  be  a  misnomer,  as  ptomaines 
are  sometimes  produced  from  vegetable  substances  in 
the  process  of  decomposition ;  they  suggest  the  term 
" putrefactive  alkaloids"  as  preferable.  The  definition 
of  a  ptomaine  given  by  these  observers  is  ua  chemical 
compound,  basic  in  character,  formed  by  the  action  of 
bacteria  on  organic  matter."  The  chemistry  of  these 
bodies  is  very  complex,  and  for  a  satisfactory  description 
of  them  Vaughan  and  Novy's  book1  is  brief  and  excel- 
lent. Among  the  ptomaines  the  following  appear  to 
be  important:  Methylamin  (CH3NH2),  the  simplest  or- 
ganic base  formed  in  the  process  of  putrefaction;  dime- 
thylamin  ((CH3)2NH) ;  trimethylamin  (C3H9N  =  (CH3)3N) ; 
ethylamin  (C2H5.NH2);  diethylamin  (C4HUN  ==  (C2H5)2- 
NH) ;  triethylamin  (C6H15N  ==  (C2H5)3N) ;  propylamin 
(C3H7.NH2) ;  butylamin  C4HUN) ;  iso-amylamin  ;  caproyl- 
amin  ;  tetanotoxin  ;  spasmotoxin  ;  dihydrolutidin  ;  putres- 
cin  ;  cadaverin  ;  neuridin  ;  saprin  ;  pyocyanin  ;  and  tyro- 
toxicon.  It  is  supposed  that  the  cases  of  ice-cream  and 
cheese-poisoning  that  sometimes  occur  are  due  to  tyro- 
toxicon  produced  by  the  putrefaction  of  the  proteid  sub- 
stances of  the  milk  before  it  is  frozen  into  ice  cream  or 
made  into  cheese.  The  safeguard  is  to  freeze  the  milk 
only  when  perfectly  fresh  and  avoid  adding  the  sugar  and 
flavoring  substances,  allowing  the  whole  to  stand  some 
time,  and  then  freezing.  Numerous  others  have  been 
described,  some  toxic,  others  harmless. 

It  is  to  compounds  of  this  kind  that  the  occasional 
cases  of  "  Fleishvergiftung "   or  "meat-poisoning"  are 

1  Ptomaines  and  Lcucomalnes. 


52  PATHOGENIC  BACTERIA. 

due,  the  growth  of  various  bacteria  in  stale  meat  bring- 
ing'about  in  its  proteid  substances  the  development  of 
toxic  ptomaines.  Kaensche1  carefully  investigated  the 
subject,  and  gives  a  synoptical  table  containing  all  the 
bacteria  of  this  class  described.  His  researches  show 
that  there  are  at  least  three  different  bacilli  whose  growth 
causes  the  development  of  poisonous  ptomaines  in  meat. 

Toxins  and  toxalbumins  are  also  very  common. 

3.  Chromogenesis.—^\MOS^  bacteria  which  produce  col- 
ored colonies  or  impart  color  to  the  medium  in  which 
they  grow  are  called  chromogenic ;  those  with  which  no 
color  is  associated,  non-chromogenic.  Most  chromogenic 
bacteria  are  saprophytic  and  non-pathogenic.  Some  of 
the  pathogenic  forms,  as  the  Staphylococcus  pyogenes 
aureus  and  citreus,  are,  however,  color-producers.  It 
seems  likely  that  the  bacteria  do  not  form  the  actual 
pigments,  but  certain  chromogenetic  substances  which, 
uniting  with  substances  in  the  culture-medium,  pro- 
duce the  colors. 

Galleotti  has  described  two  kinds  of  pigment,  one  of 
which,  being  soluble,  readily  penetrates  all  neighboring 
portions  of  the  culture-medium,  like  the  colors  of  Bacillus 
pyocyaneus,  and  an  insoluble  pigment  which  does  not 
tinge  the  solid  culture-media  at  all,  but  is  constantly 
found  associated  with  the  colonies,  like  the  pigment  of 
Bacillus  prodigiosus.  The  pigments  are  found  in  their 
greatest  intensity  near  the  surface  of  the  colony.  The 
coloring  matter  never  occupies  the  protoplasm  of  the 
bacteria  (except  the  Bacillus  prodigiosus,  in  whose  cells 
occasional  pigment-granules  may  be  seen),  but  occurs  in 
an  intercellular  excrementitious  substance. 

The  pigments  are  so  varied  as  to  give  almost  every 
known  color.  It  sometimes  happens  that  a  bacterium 
will  elaborate  two  or  more  colors.  The  Bacillus  pyo- 
ryuneus  thus  produces  pyocyanin  and  fluorescin,  both 
being  soluble  pigments — one  blue,  the  other  green. 
Gessanl  has  shown  that  when  the  Bacillus  pyocyaneus 

1  Zeitsch rift fii r  //I-N/.-//  .  etc.,  IJ<1.  xxii.,  Heft  I,  June  25,  1896. 


BIOLOGY  OF  BACTERIA.  53 

is  cultivated  upon  white  of  egg,  it  produces  only  the 
green  fluorescent  pigment,  while  in  pure  peptone  solu- 
tion it  grows  with  the  production  of  blue  pyocyanin 
alone.  His  experiments  prove  a  very  interesting  fact, 
that  for  the  production  of  fluorescin  it  is  necessary  that 
the  culture-medium  contain  a  definite  amount  of  a 
phosphatic  salt.  Sometimes  one  pigment  is  soluble, 
the  other  insoluble,  so  that  the  colony  will  appear  one 
color,  the  medium  upon  which  it  grows  another.  Some 
organisms  will  only  produce  their  colors  in  the  light ; 
others,  as  the  Bacillus  mycoides  roseus,  only  in  the  dark. 
Some  produce  them  only  at  the  room-temperature,  but, 
though  growing  luxuriantly  in  the  incubator,  refuse  to 
produce  pigments  at  so  high  a  temperature.  Thus, 
Bacillus  prodigiosus  produces  a  brilliant  red  color  when 
growing  at  the  temperature  of  the  room,  but  is  colorless 
when  grown  in  the  incubator.  Colored  lights  seem  to 
have  no  modifying  influence  upon  the  pigment-produc- 
tion. Even  if  for  successive  generations  the  bacterium 
be  grown  so  as  to  be  colorless,  it  speedily  recovers  its 
primitive  color  when  restored  to  its  old  environment,  no 
matter  what  the  color  of  the  light  thrown  upon  it.  Bac- 
teria which  have  been  robbed  of  their  color  by  incuba- 
tion, when  placed  in  the  normal  environment  produce 
the  original  color,  no  matter  what  color  the  light  they 
receive.  Some  of  the  pigments— perhaps  most  of  them — 
are  formed  only  in  the  presence  of  oxygen. 

4.  Liquefaction  of  Gelatin. — When  certain  forms  of 
bacteria  are  grown  in  gelatin  the  culture-medium  is 
partly  or  entirely  liquefied.  This  characteristic  is  en- 
tirely independent  of  any  other  property  of  the  bacte- 
rium, and  is  one  manifested  alike  by  pathogenic  and 
non-pathogenic  individuals.  Sternberg  and  Bitter  have 
shown  that  if  from  a  culture  in  which  liquefaction  has 
taken  place  the  bacteria  be  removed  by  filtration,  the 
filtrate  will  retain  the  power  of  liquefying  gelatin,  show- 
ing that  the  property  is  not  resident  in  the  bacteria,  but 
in  some  substance  in  solution  in  their  excreted  products. 


54  PA  THOGENIC  BACTERIA . 

These  products  are  described  as  "tryptic  enzymes"  by 
Fermi,  who  found  that  heat  destroyed  them.  Mineral 
acids  seem  to  check  their  power  to  act  upon  gelatin. 
Formalin  renders  the  gelatin  insoluble.  As  some  of 
the  bacteria  not  only  liquefy  the  gelatin,  but  do  so  in  a 
peculiar  and  constantly  similar  manner,  the  presence  or 
absence  of  the  change  becomes  extremely  useful  for  the 
separation  of  different  species. 

5.  Production  of  Acids  and  Alkalies.—  Under  the  head 
of  "Fermentation"  the  formation  of  acetic,  lactic,  and 
butyric  acids  has  been  discussed.     These,  however,  are 
by  no  means  all  the  acids  resulting  from  microbic  me- 
tabolism.    Ziegler  mentions  formic,  propionic,  baldrianic, 
palmitic,  and  margaric  as  being  among  those  produced, 
and  even  this  list  may  not  comprehend  them  all.     As 
the  acidity  due  to  the  microbic  metabolism  progresses,  it 
impedes,  and  ultimately  completely  inhibits,  the  develop- 
ment of  the  bacteria.     The  addition  of  phenolphthalein 
and   litmus  to  the   culture-medium  is  one  of  the  best 
methods  for  detecting  the  acids.     Milk,  to  which  litmus 
is  added,  is  particularly  convenient.     Rosalie  acid  may 
also  be  used,  the  acid  converting  its  red  into  an  orange 
color.     The  same  tests  will  also  determine  the  alkali- 
production,  which  occurs  rather  less  frequently  than  acid- 
formation  and  depends  chiefly  upon  the  salts  of  ammo- 
nium. 

6.  Production  of  Gases. — This  seems,  in  reality,  to  be 
a  part  of  the  process  of  decomposition  and  fermentation. 
Among  the  gases  due  to  bacterial  action,  CO2,  H2S,  NH4, 
CH4,  and  others  have  been  described.     If  the  bacterium 
be  anaerobic  and  develop  at  the  lower  part  of  a  tube  of 
gelatin,  not  infrequently  a  bubble  of  gas  will  be  formed 
about  the  colonies.     This  is  almost  constant  in  tetanus 
and    malignant   edema.     Ordinarily,    the  production    or 
liberation  of  gases  passes  undetected,  the  vapors  escaping 
fn.ni  ilu-  surface  of  the  culture-medium. 

To  determine  the  gas  production  where  it  is  suspected 
but  not  apparent,  the  ordinary  fermentation-tubes  can  be 


BIOLOGY  OF  BACTERIA.  55 

employed.  They  are  filled  with  glucose  bouillon,  steril- 
ized as  usual,  inoculated  and  allowed  to  grow.  If  gases 
are  formed,  the  bubbles  ascend  and  the 
gas  accumulates  at  the  top  of  the  tube. 
In  estimating  quantitatively,  one  must 
be  careful  that  the  tube  is  not  so  con- 
structed as  to  allow  the  gas  to  escape  as 
well  as  to  ascend  in  the  main  reservoir. 
For  the  determination  of  the  nature 
of  the  gases  ordinarily  produced,  some 
of  which  are  inflammable  and  some  not, 

mi        i_    u    r»      '^i     i  11  FIG.  5. — Smith's  fer- 

Theobald  Smith  has  recommended  the        raentation-tube. 
following  methods: 

"The  bulb  is  completely  filled  with  a  2  per  cent,  so- 
lution of  sodium  hydroxid  (NaOH)  and  tightly  closed 
with  the  thumb.  The  fluid  is  shaken  thoroughly  with 
the  gas  and  allowed  to  flow  back  and  forth  from  the  bulb 
to  closed  branch,  and  the  reverse  several  times  to  insure 
intimate  contact  of  the  CO2  with  the  alkali.  Lastly, 
before  removing  the  thumb  all  the  gas  is  allowed  to  col- 
lect in  the  closed  branch  so  that  none  may  escape  when 
the  thumb  is  removed.  If  CO2  be  present,  a  partial 
vacuum  in  the  closed  branch  causes  the  fluid  to  rise  sud- 
denly when  the  thumb  is  removed.  After  allowing  the 
layer  of  foam  to  subside  somewhat  the  space  occupied  by 
gas  is  again  measured,  and  the  difference  between  this 
amount  and  that  measured  before  shaking  with  the 
sodium  hydroxid  solution  gives  the  proportion  of  CO2 
absorbed.  The  explosive  character  of  the  residue  is 
determined  as  follows:  "The  cotton  plug  is  replaced  and 
the  gas  from  the  closed  branch  is  allowed  to  flow  into  the 
bulb  and  mix  with  the  air  there  present.  The  plug  is 
then  removed  and  a  lighted  match  inserted  into  the 
mouth  of  the  bulb.  The  intensity  of  the  explosion  varies 
with  the  amount  of  air  present  in  the  bulb." 

7.  Production  of  Odors. — Of  course,  such  gases  as  H2S 
and  NH2  are  sufficiently  characteristic  to  be  described  as 
odors.  There  are,  however,  a  considerable  number  of 


56  PATHOGENIC  BACTERIA. 

pungent  oders  which  seem  dependent  purely  upon  odor- 
iferous principles  dissociated  from  gases.  Many  of  them 
are  extremely  unpleasant,  as  the  onion-like  odor  of  the 
tetanus  bacillus.  The  odor  does  not  have  any  direct  rela- 
tion to  decomposition,  but,  like  the  colors  and  acids, 
seems  to  be  a  peculiar  individual  characteristic  of  the 
metabolism  of  the  organism. 

8.  Production   of  Phosphorescence. — A  Bacillus   phos- 
phorescens  and  numerous  other  organisms   have  a  dis- 
tinct phosphorescence  associated  with  their  growth.     It 
is  said  that  so  much  illumination  is  sometimes  caused  by 
a  gelatin  culture  of  some  of  these  as  to  enable  one  to  tell 
the  time  by  a  watch.     Most  of  them  are  found  in  sea- 
water,  and  are  best  grown  in  sea-water  gelatin. 

9.  frodnclion  of  Aromatics. — The  most  important  of 
these  is  indol,  which  was  at  one  time  thought  to  be  pecu- 
liar to  the  cholera  spirillum.     For  the  method  of  deter- 
mining its  presence,  see  "Dunham's  Solution."    At  pres- 
ent we  know  that  a  variety  of  organisms  produce  it,  and 
that  it  and  phenol,  kresol,  hydrochinon,  hydroparacumaric 
acid,  and  paroxy-phenylic-acetic  acid  are  by  no  means 
uncommon. 

10.  Reduction  of  Nitrites. — A  considerable  number  of 
bacteria  are  able  to  reduce  nitrites  present  in  the  soil  or 
in  culture-media  prepared  for  them  into  ammonia  and 
nitrogen.     To  the  horticulturist  this  is  a  matter  of  much 
interest.     Winogradsky  has   found  a  specific   nitrifying 
bacillus  in  soil,  and  asserts  that  the  presence  of  ordinary 
bacteria  in  the  soil  causes  the  reduction  of  no  nitrites  so 
long  as  his  special  bacillus  is  withheld. 

1 1.  Peptonixation  of  Milk. — Numerous  bacteria  possess 
the  power  of  digesting—  peptonizing— the  casein  of  milk. 
The  process  differs  with  different  bacteria,  some  digesting 
the  casein  without  any  apparent  change  in  the  milk, 
smiie  producing  coagulation,  some  gelatinization  of  the 
fluid.      In   sonic  cases   the  digestion  of  the  casein  is  so 
complete  as  to  transform  the  milk  into  a  transparent 
watery  fluid. 


BIOLOGY  OF  BACTERIA.  57 

Milk  usually  contains  bacteria,  entering  it  from  the 
dust  of  the  dairy,  possessing  this  power.  In  the  process 
of  peptonization  the  milk  may  become  bitter,  but  need 
not  change  its  original  reaction.  As  the  peptonization 
progresses  the  milk  very  often  becomes  poisonous,  espe- 
cially to  individuals  under  two  years  of  age,  and  may 
bring  about  a  fatal  enterocolitis  or  "summer  complaint." 
The  disease  does  not  only  occur  in  consequence  of  toxic 
substances  formed  from  the  split-up  albumins,  or  from 
the  presence  of  metabolic  products  of  the  bacteria,  but, 
as  Liibbert  has  shown,1  from  the  presence  of  the  bacteria 
themselves.  One  reason  that  the  enterocolitis  caused  in 
this  way  comes  on  in  summer  is  that  it  is  only  in  un- 
usually warm  weather  that  these  bacteria  are  able  to 
grow  luxuriantly. 

Sometimes  the  properties  of  coagulation  and  digestion 
of  milk  are  valuable  aids  in  the  separation  of  different 
species  of  bacteria. 

12.  Production  of  Disease. — Bacteria  which  produce 
diseases  are  known  as  pathogenic;  those  which  do  not, 
as  non-pathogenic.  Between  the  two  groups  there  is  no 
sharp  line  of  separation,  for  true  pathogens  may  be  culti- 
vated under  such  adverse  conditions  that  their  virulence 
will  be  entirely  lost,  while  at  times  bacteria  ordinarily 
harmless  may  be  made  toxic  by  certain  manipulations  or 
by  introducing  them  into  animals  in  certain  combina- 
tions. The  diseases  produced  are  the  result  of  the  sum 
of  numerous  activities  exhibited  by  the  bacteria.  For 
example,  it  may  be  that  a  microbe,  having  effected  its 
entrance  into  an  animal,  grows  with  great  rapidity, 
completely  blocking  up  the  blood-  and  lymph-channels, 
so  that  the  proper  circulation  of  these  fluids  is  stopped 
and  disease  and  death  must  result.  Perhaps  more  com- 
mon than  this  is  a  local  establishment  of  the  organisms, 
with  a  resulting  inflammation,  due  partly  to  the  presence 
of  the  foreign  organisms,  and  partly  to  their  toxic  me- 
tabolic products.  More  often,  however,  the  pathogenic 

1  Zeitschrift  fur  Hygiene,  xxii.,  Heft  2,  1896,  p.  I. 


58  PA  THOGNEIC  BA  CTERIA. 

bacteria  produce  powerful  metabolic  poisons — toxins, 
ptomaines,  etc. — which  either  cause  widespread  destruc- 
tion of  the  tissues  immediately  acted  upon,  or,  circulating 
throughout  the  organism,  produce  fever,  nervous  excita- 
tion, and  a  general  overthrow  of  the  normal  physiological 
equilibrium.  These  peculiarities  serve  to  divide  the  bac- 
teria into 

Phlogistic  bacteria, 

Toxic  bacteria, 
Septic  bacteria. 

The  bacteria  of  suppuration  probably  act  in  several 
ways.  Their  products  may  be  of  a  violently  chemotactic 
nature,  or  their  virulence,  exerted  upon  the  surrounding 
tissue,  may  destroy  large  numbers  of  the  cells,  whose 
dead  bodies  may  be  chemotactic.  When  the  suppura- 
tion is  violent  the  toxic  product  of  the  bacterium  is  itself 
most  probably  strongly  chemotactic. 

The  great  majority  of  suppurations  depend  upon  bac- 
teria, but  there  are  sterile  suppurations  which  sometimes 
follow  the  use  of  croton  oil,  turpentine,  etc.  The  differ- 
ence between  infectious  and  sterile  pus  is  marked,  for 
the  former,  containing  the  virulent  germs,  tends  to  invade 
new  tissue  or  distribute  its  disease-producers  to  new  parts 
of  the  body,  while  the  latter  remains  local. 

There  are  few  purely  toxic  bacteria,  the  tetanus  and 
diphtheria  bacilli  serving  as  typical  examples.  By  sep- 
tic bacteria,  I  mean  those  whose  habitual  tendency  is  to 
grow  in  the  blood  and  lymph  and  distribute  to  all  the 
organs.  Anthrax  is  a  type  of  the  class. 

How  the  disease-producing  bacteria  effect  their  en- 
trance into  the  tissues  is  an  interesting  question.  The 
channels  naturally  open  to  them  are  those  leading  into 
the  interior  of  the  organism,  and  must  be  separately  con- 
sidered. 

(//)  ////  /Vs',  stive  Tract.—  Attention  has  already  been 
called  to  the  facility  with  which  the  bacteria  enter  the 
digestive  tract  in  foods  and  drinks.  Once  their  metabo- 
lism i-  in  active  progress,  the  poisons  which  they  produce 


BIOLOGY  OF  BACTERIA.  59 

are  ready  for  absorption.  It  seems  probable  that  the 
absorption  of  the  toxic  substances  by  reducing  the  vital- 
ity of  the  individual  predisposes  to  the  formation  of  local 
lesions  through  which  the  bacteria  may 'enter  the  intes- 
tinal walls  to  continue  their  existence  and  produce 
greater  damage  than  before.  Some  such  theory  may 
explain  the  activity  of  such  organisms  as  those  of 
typhoid,  cholera,  and  meat-poisoning,  but  it  is  not  true 
that  all  bacteria  can  be  admitted  into  the  intestinal  struc- 
ture in  this  way,  for  the  experiments  of  Max  Neisser,1  who 
fed  mice,  guinea-pigs,  and  rabbits  upon  a  variety  of 
pathogenic  and  non-pathogenic  bacteria,  both  before  and 
after  injuries  to  the  intestine  caused  by  the  ingestion  of 
powdered  glass,  chemical  agents,  and  irritating  bacteria, 
failed  to  show  tliat  with  the  exception  of  those  bacteria 
whose  particular  tendency  is  toward  the  production  of 
intestinal  disease,  none  entered  either  the  chyliferous 
system,  the  blood-vessels,  or  the  organs. 

The  occurrence  of  the  staphylococcus  aureus  and  other 
bacteria  in  osteomyelitis,  and  of  tubercle  bacilli  in  deep- 
seated  diseases  of  the  bones  and  internal  organs,  has  led 
many  to  believe  that  the  intestine  is  a  point  of  easy 
entrance.  There  is,  however,  no  reason  to  believe  that 
penetration  of  the  digestive  mucous  membrane  is  any 
easier  than  that  of  the  respiratory  or  other  similarly  deli- 
cate tissues. 

On  the  other  hand,  Beco 2  is  of  the  opinion  that,  with- 
out any  apparent  lesion  of  the  intestine,  bacteria— ba- 
cillus coli — escape  from  it  into  the  blood  during  life. 
His  experiments  showed  that  immediately  after  death  the 
colon  bacillus  could  be  found  in  small  numbers  in  the 
spleen,  in  many  cases.  After  twenty-four  hours,  in 
three  cases,  they  were  present  in  immense  numbers. 
When,  however,  they  were  absent  from  the  organ  im- 
mediately after  death,  they  were  also  absent  after  twenty- 
four  hours. 

1  Zeitschrift  fur  Hygiene,  June  25,  1896,  Bd.  xxii.,  Heft  I. 

2  Ann.  de  rinst.  Pasteur,  1895,  No.  3. 


60  PA  THOGENIC  BA  CTERIA . 

Achard '  studied  49  cases  to  determine  whether  or  not 
the  intestinal  bacteria  entered  the  organism  during  the 
death  agony.  In  14  bacteria  were  found  intra  vitam  in 
the  liver  and  in  the  blood.  In  24  no  bacteria  were  found 
during  life,  but  after  death.  In  n  no  bacteria  were 
found  either  during  life  or  after  death — before  twenty-two 
to  twenty-seven  hours,  when  his  autopsies  were  made. 
The  passage  of  bacteria  into  the  blood  during  agony  was 
unusual.  The  bacteria  most  commonly  found  during  life 
were  the  streptococci  and  staphylococci.  In  the  dead  body 
the  one  most  frequently  encountered  was  the  bacillus  coli 
commnnis.  Before  reaching  the  intestine  the  bacteria 
pass  through  the  stomach,  and  must  resist  the  deleterious 
action  of  the  acid  gastric  juice,  which  few  are  able  to  do. 

(b)  The  Respiratory  Tract. — Notwithstanding  the  moist 
interiors  of  the  mouth  and  nose  and  the  lashing  cilia  of 
the  pharyngeal  and  tracheal  mucous  membrane,  numbers 
of  bacteria  enter  the  smaller  bronchioles,  and  occasionally 
penetrate  as  deeply  as  the  air-cells.  It  is  usual  to  find  a 
few  bacteria  in  a  section  of  healthy  lung. 

Thomson  and  Hewlett2  estimate  that  from  1500  to 
14,000  bacteria  are  inspired  every  hour.  As  expired  air 
is  usually  sterile,  they  sought  to  determine  what  became 
of  these  organisms,  and  agree  with  Lister  and  with 
Hildebrandt  that  the  organisms  are  arrested  before  they 
reach  the  air-cells.  They  found  by  killing  a  number  of 
animals  and  examining  the  tracheal  surface  that  it  was 
sterile,  and  conclude  that  the  great  majority  of  bacteria 
are  stopped  in  the  nose  against  the  moist  surfaces  of  its 
vestibules,  where  they  found  great  numbers  in  the  crusts. 
No  doubt  the  ciliated  cells  of  the  nose  have  something  to 
do  with  getting  rid  of  the  bacteria. 

An  ingenious  experiment  was  performed  by  placing 
some  bacilli  prodigiosus  upon  the  septum  naris,  and 
making  a  culture  from  the  spot  at  intervals  during  two 

1  Archives  de  m&lecine  exp^rimentale  et  d'antnmie  |>:itli(>lo^i<|ue,  1895,  No. 
l,  p.  25- 

1  British  Med.  Jour.,  Jan.  1 8,  1896,  p.  137. 


BIOLOGY  OF  BACTERIA.  61 

hours.  Cultures  made  within  five  minutes  showed  con- 
fluent colonies  of  the  bacilli,  which  became  fewer  and 
fewer  in  number,  until  after  two  hours  not  a  trace  of  a 
bacillus  prodigiosus  could  be  found. 

Wurtz  and  L,ermoyez  assert  that  the  nasal  mucus  exerts 
a  germicidal  action,  but  this  is  not  substantiated.  These 
writers  conclude  that  the  bacteria  were  carried  away  by 
the  action  of  the  cilia  and  trickling  mucus. 

It  seems  to  have  been  proven  by  Buchner  that  micro- 
organismal  infection  may  take  place  through  the  lungs 
without  definite  breach  of  continuity  of  the  alveolar 
walls.  He  mixed  anthrax  spores  and  lycopodium  powder 
together,  and  caused  mice  and  guinea-pigs  to  inhale  them. 
Out  of  the  66  animals  used  in  his  experiments,  50  died  of 
anthrax  and  9  of  pneumonia.  Our  knowledge  of  the  dis- 
position of  foreign  particles  in  the  lung  probably  explains 
such  infection  by  assuming  that  the  presence  of  the  lyco- 
podium attracted  numerous  leucocytes  to  the  affected  air- 
cells;  that  these  took  up  the  powder,  and  with  it  the 
spores;  and  that  the  leucocytes,  being  cells  of  very  sus- 
ceptible animals,  were  unable  to  resist  the  growth  into 
bacilli  of  the  spores  which  they  had  carried  into  the 
lymph-channels. 

On  the  other  hand,  it  has  been  shown  that  when 
the  entering  spores  are  unaccompanied  by  a  mechanical 
irritant  like  the  lycopodium  powder,  but  are  inspired 
in  a  pulverized  liquid,  infection  takes  place  much  less 
readily. 

Tuberculosis  and  pneumonia  are  in  all  probability 
generally  the  result  of  the  inspiration  of  the  specific 
organisms. 

(c)  The  Skin  and  the  Superficial  Mucous  Membranes. — 
The  entrance  of  bacteria  into  the  tissues  by  way  of  the 
skin  is  probably  extremely  rare  if  the  skin  is  sound. 
Numerous  experimenters  have  caused  infection  by  rub- 
bing bacteria  or  their  spores  upon  the  skin.  It  would 
seem  probable  that  in  these  cases  there  must  have 
been  some  microscopic  lesions  into  which  the  bacteria 


62  PA  THOGENIC  BA  CTERIA . 

were  forced.  My  own  investigations  have  shown  viru- 
lent staphylococci  of  suppuration  upon  the  conjunctivae 
in  health.  It  is  very  improbable  that  the  bacteria  habit- 
ually present  upon  the  skin,  and  ready  to  enter  the  least 
abrasion,  can  penetrate  the  outer  coverings  of  the  body, 
except  when  disease  or  accident  has  rendered  them 
abnormally  thin  or  macerated. 

Turro  seems  to  have  shown  that  the  gonococcus  can 
enter  the  tissues  without  any  pre-existing  lesion,  for  he 
asserts  that  if  a  virulent  culture  simply  be  touched  to 
the  meatus  urinarius,  the  disease  will  be  established. 

(d)  Wounds. — The  results  of  the  entrance  of  bacteria 
into  unprotected  wounds  are  now  so   familiar  that  no 
one  deserving  of  the  name  of  surgeon  dares  to  allow  a 
wound  to  go  undressed. 

(e)  The  Placenta. — Very  frequently  the  occurrence  of 
specific   diseases   during   pregnancy  causes   abortion   of 
the  product  of  conception.     In  certain  cases  the  specific 
contagion  passes  through  the  placenta  and  infects  the 
fetus.     This  has   been   pretty  clearly  demonstrated   for 
variola,  malaria,  syphilis,  measles,  anthrax,  symptomatic 
anthrax,  glanders,  relapsing  fever,  typhoid,  and  in  rare 
cases  for  tuberculosis. 

Anche  found  streptococci  and  staphylococci  in  the  tis- 
sues of  aborted  foeti  in  cases  of  variola.1  Except  in  the 
case  of  wounds,  it  must  be  observed  that,  although  the 
bacteria  are  in  the  body—/.  <?.,  respiratory,  digestive,  or 
sexual  apparatus,  etc.— they  are  still  not  in  the  blood,  and 
really  not  in,  but  only  upon  the  surfaces  of  the  tissues. 

For  their  actual  entrance  into  the  circulation,  Kruse2 
gives  the  following  possible  modes: 

1.  Passive  entrance  of  the  bacteria  through  the  sto- 
mata  of  the  vessels  where  the  pressure  of  the  inflammatory 
exudate  is  greater  than  the  intravascular  pressure. 

2.  Entrance  of  the  bacteria  into  the  vessel  in  the  body 
of  leucocytes  that  have  incorporated  them. 

1  La  Semaine  Mid.,  1892,  No.  6l. 
«  Fltigge's  Mikroorganismen. 


BIOLOGY  OF  BACTERIA.  63 

3.  Actual  penetration  of  the  vessel-wall  by  the  growth 
of  the  microorganism. 

4.  Entrance  into  the  vessels  via  the  lymphatics,  either 
passively  or  in  leucocytes. 

Seeing  that  the  channels  by  which  bacteria  can  enter 
the  body  are  so  numerous,  and  that  there  'is  scarce  a 
moment  when  some  part  of  us  is  not  in  contact  with 
them,  how  is  it  that  we  are  not  constantly  subject  to 
disease  ?  The  consideration  of  this  question,  together 
with  the  closely  related  questions  why  we  should  be 
subject  to  certain  diseases  only,  and  to  these  diseases 
at  certain  times  only,  must  be  reserved  for  another  chap- 
ter, in  which  the  subjects  Immunity  and  Susceptibility  can 
be  taken  up  at  length.  Before  passing  on  to  it,  however, 
some  attention  must  be  paid  to  the  subject  of  the 

Elimination  of  Bacteria  from  the  Body. — There  is  every 
reason  to  think  that  non-pathogenic  bacteria  entering  the 
body  ordinarily,  or  being  experimentally  injected  into  it, 
follow  the  same  course  as  inert,  non-vital  particles;  con- 
cerning which,  the  experiments  of  Siebel  have  shown 
that  they  accumulate  in  the  finest  capillaries,  especially 
in  the  lung,  liver,  spleen,  and  bone-marrow,  and  are 
slowly  transferred  to  the  surrounding  tissues,  either  to  be 
collected  in  the  connective-tissues,  carried  to  the  lym- 
phatic nodes,  or  to  be  subsequently  excreted  with  the 
bile,  succus  entericus,  etc. ,  or  to  be  discharged  from  the 
surface  of  the  mucous  membranes,  pulmonary  alveoli, 
tonsils,  etc.  They  also  escape  from  suppurating  wounds 
to  which  they  may  be  carried  by  leucocytes.  They  are 
not  excreted  by  the  kidneys. 

The  experiments  of  Wyssokowitsch  are  in  accord  with 
the  results  of  Siebel' s  work,  and  show  that  the  kidney 
rarely  eliminates  bacteria.  Cavazzani  found  that  the 
kidney  had  the  power  to  retain  bacteria  in  the  blood, 
unless  the  epithelium  was  injured. 

The  principal  avenues  of  escape  for  the  bacteria  are, 
therefore,  for  the  non-pathogenic  forms,  the  mucous  mem- 
branes, the  bile,  and  the  sweat.  For  the  pathogenic 


64  PATHOGENIC  BACTERIA. 

forms,  the  mucous  membranes,  the  intestine  in  particular 
in  such  diseases  as  anthrax,  typhoid,  and  cholera;  the  bile 
almost  always;  the  sweat  generally;  the  kidney  when 
damaged;  the  mammae  in  tuberculosis  and  septicemia 
particularly,  and,  of  course,  such  of  the  pathological 
products  of  the  disease-process  as  pus  from  abscesses, 
dejecta  of  typhoid  and  cholera,  expectoration  in  diph- 
theria and  tuberculosis,  etc. 

The  bacteria  that  are  not  excreted,  but  retained  in  such 
organs  as  the  spleen,  bone-marrow,  and  lymphatic  nodes, 
are  probably  slowly  devitalized  and  dissolved. 


CHAPTER   III. 
IMMUNITY  AND  SUSCEPTIBILITY. 

ONE  of  the  most  interesting  things  observed  in  physi- 
ology and  pathology  is  the  resistance  which  certain  ani- 
mals show  to  the  invasion  of  their  bodies  by  the  germs 
of  disease. 

Thus,  man  suffers  from  typhoid  fever,  cholera,  and 
other  infectious  diseases  which  are  never  observed  in  the 
domestic  animals;  cattle  are  subject  to  a  pleuro-pneumo- 
nia  which  does  not  affect  their  attendants;  man,  the  cow, 
and  the  guinea-pig  are  peculiarly  susceptible  to  tubercu- 
losis, which  the  cat,  dog,  and  horse  resist;  yellow  fever 
is  a  highly  contagious,  infectious  disease  which  is  almost 
certain  to  attack  all  new  arrivals  of  the  human  species 
when  epidemic,  but  which  rarely,  if  ever,  attacks  animals. 

The  popular  mind  accepts  the  statement  of  such  facts 
as  these  without  any  other  explanation  than  that  the 
animals  are  different,  and  so  of  course  their  diseases  are 
different;  but  the  more  the  scientific  man  contemplates 
them,  the  more  complicated  the  matter  becomes;  for, 
while  it  might  be  admitted  that  a  difference  -in  the  body- 
temperature  and  chemistry  might  explain  why  a  frog 
will  resist  anthrax,  which  readily  kills  a  white  mouse,  it 
will  not  explain  why  a  house-mouse,  whose  chemistry 
must  be  almost  identical  with  that  of  the  white  mouse, 
can  successfully  combat  the  disease.  Nor  is  this  all. 
That  one  attack  of  yellow  fever,  of  typhoid  fever,  or 
of  scarlet  fever  renders  a  second  attack  almost  impos- 
sible is  not  the  less  interesting  because  of  its  every-day 
observation.  The  mouse  that  has  recovered  from  teta- 
nus will  not  take  tetanus  again,  and  most  interesting  and 


66  PATHOGENIC  BACTERIA. 

extraordinary  is  the  fact  that  a  few  drops  of  blood  from 
the  recovered  mouse  injected  into  another  will  protect  it 
from  tetanus. 

Imnninitv  is  the  condition  in  which  the  body  of  an 
animal  resists  the  entrance  of  disease-producing  germs, 
or,  having  been  compelled  to  allow  them  to  enter,  resists 
their  growth  and  pathogenesis.  The  resistance  so  mani- 
fested is  a  distinct,  potential  vital  phenomenon. 

Susceptibility  is  the  opposite  condition,  in  which,  in- 
stead of  resistance,  there  is  a  passive  inertia  which  allows 
the  disease-producing  organisms  to  develop  without  oppo- 
sition. Susceptibility  is  accordingly  the  absence  of  im- 
munity. 

Immunity  is  either  natural  or  acquired. 

Natural  Immunity.— By  this  term  is  meant  the  natural 
and  constant  resistance  which  certain  healthy  animals 
exhibit  toward  certain  diseases. 

The  white  rat  is  peculiar  in  resisting  anthrax.  It  is 
almost  impossible  to  develop  anthrax  in  a  healthy  white 
rat,  but  Roger  found  that  such  an  animal  would  easily 
succumb  to  the  disease  if  compelled  to  turn  a  revolving 
wheel  until  exhausted.  Susceptibility  which  follows  such 
an  exhaustion  of  the  vital  powers  cannot  be  regarded  as 
other  than  accidental,  and  makes  no  exception  to  the 
statement  that  the  white  rat  is  immune  to  anthrax. 
Animals  such  as  man,  sheep,  cows,  rabbits,  and  white 
mice  are  susceptible  to  anthrax,  while  birds  and  reptiles 
are  generally  immune.  The  great  difference  in  the  morph- 
ology between  mammals  and  birds  and  reptiles,  together 
with  the  fact  that  their  temperature,  blood,  and  tissues 
all  differ,  makes  this  immunity  reasonably  intelligible. 
Morphological  differences,  however,  will  not  suffice  to 
explain  all  cases,  for  the  Caucasian  nearly  always  suc- 
cumbs to  yellow  fever,  while  the  negro  is  rarely  affected  ; 
and  scarlatina,  which  is  one  of  our  commonest  and  most 
us  diseases  of  childhood,  is  said  to  be  unknown 
the  Japanese.  Nor  is  this  all,  for,  close  as  is  their 
resemblance  in  all  respects  except  color,  the  house-mouse, 


IMMUNITY  AND  SUSCEPTIBILITY.  67 

field-mouse,  and  white  mouse  differ  very  much  in  their 
susceptibility  to  various  diseases. 

Acquired  immunity  is  resistance  which  is  the  result 
of  accidental  circumstances.     It  may  result — 

A.  By  recovery  from  a  mild   attack  of  the   disease. 
Most  adults  have  suffered  from  rubeola,  scarlatina,  and 
varicella  in  childhood,  and  in  consequence  of  the  attacks 
are  now  immune  to  these  diseases — i.  e.  will  not  become 
affected  again.     One  attack  of  yellow  fever  is  always  a 
complete  guard  against  another.    Typhoid  fever  is  rarely 
followed 'by  a  second  attack. 

B.  By  recovery  from  an  attack  of  a  slightly  different 
disease.     Sometimes  the  immunity  is  experimentally  pro- 
duced, as  when  by  vaccination  we  produce  the  vaccine 
disease  and  afterward  resist  variola.    Acquired  immunity 
is  a  little  less  complete  and  not  so  permanent  as  natural 
immunity,  for  in  the  latter  it  is  only  when  the  functions 
of  the  individual  are  disturbed  or  his  vitality  depressed 
that  the  resistance  is  lost,  while  in  the  former  time  seems 
to  lessen  the  power  of  resistance,   so  that  rubeola  and 
scarlatina  may  return  in  a  few  months  or  years,  and  for 
complete  protection  vaccination  may  need  to  be  done  as 
often  as  every  seven  years. 

C.  By    the    injection   of    antitoxic    substances.      At 
present    there   is    much    agitation    over   the    newly-dis- 
covered antitoxin  of  diphtheria,  the  injection  of  about 
500  units  of  which  will  give  complete  protection  against 
the   disease  for   a  period  lasting  from  a  month  to  six 
weeks. 

Immunity  may  be  destroyed  in  numerous  ways: 
(a)  By  variation  from  the  normal  temperature  of  the 
animal  under  observation.  Pasteur  observed  that  chick- 
ens would  not  take  anthrax,  and  suspected  that  this 
immunity  might  be  due  to  their  high  body-temperature. 
After  inoculation  he  plunged  the  birds  into  a  cold  bath, 
reduced  their  temperature,  and  succeeded  in  destroying 
their  immunity.  The  experiment  was  a  success,  but  the 
reasoning  seems  to  have  been  faulty,  as  the  sparrow, 


68  PATHOGENIC  BACTERIA. 

with  a  temperature  equally  high,  readily  falls  a  victim 
to  anthrax  without  a  cold  bath. 

(b)  By  altering  the  chemistry  of  the  blood  by  changing 
the  diet  or  by  hypodermic  injection.     Leo  found  that 
when  white  rats  were  injected  with  or  fed  upon  phlorid- 
zin  an  artificial  glycosuria  resulted  which  destroyed  their 
natural  resistance  to  anthrax.     Hankin  found  that  rats, 
which  possess  considerable  immunity  to  anthrax,  could 
be  made  susceptible  by  a  diet  of  bread.     Platania  suc- 
ceeded in  producing  anthrax  in  dogs,  frogs,  and  pigeons, 
naturally  immune,  by  subjecting  them  to  the  influence 
of  curare,  chloral,  and  alcohol. 

(c)  By  diminishing  the  strength  of  the  animal.     Roger 
by  compelling  white  rats  to  turn  a  revolving  wheel  until 
exhausted  destroyed  their  immunity  to  anthrax. 

(d)  By  removing  the  spleen  (?).      A  large  number  of 
experiments  have   been   performed   by   various   investi- 
gators to  show  that  the  removal  of  the  spleen  does  or 
does  not  affect  immunity.     From  their  work  it  seems 
proper  to  conclude  that  the  spleen  has  little,  if  any,  in- 
fluence upon  the  vital  resistance  to  disease. 

I.  Bardach,1  Righi,2and  Montuori3  seem  to  have  shown 
that  the  removal  of  the  spleen  lessens  the  ability  of  the 
organism  to  combat  the  infections. 

II.  Blumenreich  and  Jacoby,4  on  the  contrary,  found 
that  the  removal  of  the  spleen  was  followed  by  a  hyper- 
leucocytosis,  an  increase  in  the  bactericidal  power  of  the 
blood,  and  consequent  increase  of  immunity. 

III.  Milkinow-Raswedenow5  found  that  the  removal  of 
the  spleen  was  a  weakening  factor  in  the  immunization 
of  animals.     The  spleen  itself,   however,  was  of  little 
importance  in  combating  the  micro-organismal  infections. 

Kurlow6  concluded  from  his  experiments  that  the  in- 

1  Ann.Je  r/nst.  Pastfitr,  1889,  No.  2,  p.  577,  and  1891,  No.  I,  p.  40. 

AV/o/w,;  .\/f,/i,-at  1893,  PP-  17°,  171- 

»  Ibid.,  Feb.,  1893,  17,  1 8.       «  Btrlin.  klin.  Wochenschrift,  May  24,  1897. 
6  Zeitschrift  „?,  1896,  xxi.,  3. 

•Archn  .'.  INS.,,  i;,i.  ix.,  p.  450. 


IMMUNITY  AND  SUSCEPTIBILITY  69 

fluence  of  the  spleen  was  not  greater  than  that  of  any 
other  organ  in  overcoming  bacterial  infections. 

Kanthack  l  found  that  the  removal  of  the  spleen  had 
practically  no  influence  upon  the  natural  immunity  of 
animals  to  pyocyaneus  infection. 

(e)  By  combining  two  different  species  of  bacteria^  either 
of  which,  when  injected  alone,  would  be  harmless  or  of 
slight  effect.  Roger  found  that  when  animals  immune 
to  malignant  edema  were  simultaneously  injected  with 
i  to  2  c.cm.  of  a  culture  of  Bacillus  prodigiosus  and  the 
bacillus  of  malignant  edema,  they  would  contract  the 
disease.  Pawlowski  found  that  when  rabbits,  which 
are  very  susceptible  to  anthrax,  were  simultaneously  in- 
jected with  anthrax  and  prodigiosus,  they  recovered 
from  the  anthrax,  as  if  the  harmless  microbe  possessed 
the  power  of  neutralizing  the  products  of  the  patho- 
genic form. 

Giarre  found  that  if  an  adult  guinea-pig,  which  is  refrac- 
tory to  infection  by  pneumococci,  were  simultaneously  in- 
oculated with  diphtheria,  it  readily  died  of  septicemia. 

Sometimes  an  apparent  immunity  depends  upon  the 
attenuation  of  the  culture  used  for  inoculation,  and  the 
erroneous  results  to  which  such  a  mistake  may  lead  are 
obvious.  Should  a  culture  become  attenuated,  its  viru- 
lence may  sometimes  be  increased  by  inoculating  it  into 
the  most  susceptible  animal,  then  from  this  to  a  less 
susceptible,  and  then  to  an  immune  animal.  The  viru- 
lence of  anthrax  is  increased  by  inoculation  into  pigeons, 
and  also  by  cultivation  in  an  infusion  of  the  tissues  of 
an  animal  similar  to  the  one  to  be  inoculated. 

It  must  be  understood  that  the  term  "immunity"  is 
a  relative  one,  and  that  while  "a  white  rat  is  immune 
against  anthrax  in  amounts  sufficiently  large  to  kill  a 
rabbit,  it  is  perhaps  not  immune  against  a  quantity 
sufficiently  large  to  kill  an  elephant." 

It  is  not  to  be  expected  that  such  intricate  phenomena 
as  these  which  have  been  mentioned  could  be  observed 

1  Centralbl.f.  Bakt.  it.  Parasiienk.,  1892,  xii.,  p.  227. 


70  PA  THOGENIC  BA  CTERIA . 

and  suffered  to  go  unexplained.  We  have  explanations, 
but,  unfortunately,  they  are  as  intricate  as  the  phenomena, 
and,  though  each  may  possess  its  grain  of  truth,  not  one 
will  satisfy  the  demands  of  the  thoughtful  student.  In 
brief  review,  the  theories  of  immunity  are  the  following : 

1.  THE  EXHAUSTION  THEORY.— This  hypothesis  was 
advanced  by  Pasteur  in  1880,  and  suggests  that  by  its 
growth  in  the  body  the  micro-organism   uses  up  some 
substance  essential  to  its  life,  and  that  when  this  sub- 
stance is  exhausted  the  microbe  can  no  longer  thrive. 
The  removal  of  the  necessary  material,  if  complete,  will 
cause  permanent  immunity. 

As  Sternberg  points  out,  were  this  theory  true  we  must 
have  within  us  a  material  of  small-pox,  a  material  of 
measles,  a  material  of  scarlet  fever,  etc. ,  to  be  exhausted 
by  its  appropriate  organism.  It  would  necessitate  an 
almost  inconceivably  complex  body-chemistry  and  a 
rather  stable  condition  of  the  same. 

2.  THE   RETENTION   THEORY. — In    the    same    year 
Chauveau   suggested   that   the   growth   of    the   bacteria 
in  the  body  might  originate  some  substance  prejudicial 
to  their  further  and  future  development.     There  seems 
to  be  a  large  kernel  of  truth  in  this,  but  were  it  always 
the  case  we  would  have  added  to  our  blood  a  material 
of  small-pox,  a  material  of  measles,  a  material  of  scarlet 
fever,  etc.,  so  that  we  would  become  saturated  with  the 
excrementitious  products  of  the  bacteria,  instead  of  hav- 
ing so  many  substances  subtracted  from  our  chemistry. 

3.  THE  THEORY  OF  PHAGOCYTOSIS. — In  .1881,  Carl 
Roser  suggested  a  relation  between  immunity  and  the 
already   familiar   phenomenon   of  phagocytosis.     Stern- 
berg  in  the  United  States  and  Koch  in  Germany  observed 
the  sanu-  tiling,  but  little  real  attention  was  paid  to  the 
subject  until  1884,  when  Metschnikoff  appeared,  with  his 
careful  observations  upon  the  daphnia,  as  the  great  cham- 
pion of  the  theory  which  is  now  known  as  u Metschni- 
koff18  theory  of  phagocytosis." 

Phagocytosis   is   the   swallowing  or  incorporating  of 


IMMUNITY  AND  SUSCEPTIBILITY.  71 

particles  by  certain  of  the  body-cells  which  are  called 
phagocytes.  This  activity  of  the  cells  toward  inert 
particles  had  been  observed  by  Virchow  as  early  as  1840, 
and  toward  living  bacteria  by  Koch  in  1878,  but  was  not 
carefully  studied  until  1884.  Metschnikoff  divides  the 
phagocytes  into  fixed  phagocytes,  comprising  the  fixed 
connective-tissue  cells,  endothelium,  etc.,  and  the  free 
phagocytes,  which  are  the  leucocytes.  The  terms  u  phag- 
ocyte" and  "leucocyte"  are  not  to  be  regarded  as  synon- 
ymous in  this  connection  ;  all  leucocytes  are  not  phag- 
•ocytic,  the  lymphocyte  having  never  been  observed  to 
take  up  bacteria. 

It  is  obvious  that  only  those  cells  can  be  phagocytic 
which  are  without  a  resisting  cell-wall  and  possess 
ameboid  movement.  When  an  ameba,  in  a  liquid  con- 
taining numerous  diatoms  and  bacteria,  is  watched 
through  the  microscope,  an  interesting  phenomenon  is 
observed.  The  ameba  will  approach  one  of  the  vege- 
table cells,  even  though  it  may  be  at  a  distance,  will 
apprehend  and  surround  it,  and  within  the  animal  cell 
the  vegetable  cell  will  be  digested  and  assimilated.  The 
ameba  has  no  eyes,  no  nose,  no  volition,  and,  so  far  as 
we  can  determine,  no  nervous  apparatus  which  gives 
it  tactile  sense,  yet  it  will  approach  the  particle  fitted 
for  its  use  and  swallow  it.  The  attraction  which  draws 
the  cells  together  has  been  called  by  Peffer  chemotaxis, 
chemiotaxis,  or  chemotropism. 

Chemotaxis  is  the  exhibition  of  an  attractive  force 
between  cells  and  their  nutriment,  ameboid  cells  and 
food-particles,  and  sometimes  between  ameboid  cells  and 
inert  particles.  This  attractive  force,  when  operating  so 
as  to  draw  the  ameba  to  the  particle  it  will  devour,  is 
further  named  positive  chemotaxis  in  order  to  distinguish 
it  from  a  repulsive  force  sometimes  exerted  causing  the 
ameboid  cells  to  fly  from  an  enemy,  as  it  were,  and  which 
is  called  negative  chemotaxis. 

The  force  that  operates  and  guides  the  ameba  in  its 
movements  is  exactly  the  same  as  that  which  governs  the 


72  PA  THOGENIC  BA CTERIA. 

movement  of  the  phagocytic  cells  of  the  human  body, 
and  observation  of  these  phenomena  is  not  difficult.  If 
a  small  capillary  tube  be  filled  with  sweet  oil  and  placed 
beneath  the  skin,  only  a  short  time  need  pass  before  it 
will  be  found  full  of  leucocytes — positive  chemotaxis. 
If,  instead  of  sweet  oil,  oil  of  turpentine  be  used,  not 
a  leucocyte  will  be  found — negative  chemotaxis. 

Phagocytosis  is  almost  universal  in  the  micro-or- 
ganismal  diseases  at  some  stage  or  another.  If  the 
blood  of  a  patient  suffering  from  relapsing  fever  be 
studied  beneath  the  microscope,  it  will  be  found  to 
contain  numerous  active  mobile  spirilla,  all  free  in  the 
liquid  portion  of  the  blood.  As  soon  as  the  apyretic 
stage  comes  on  not  a  single  free  spirillum  can  be  found. 
Every  one  is  seen  to  be  enclosed  in  the  leucocytes. 

At  the  edge  of  an  erysipelatous  patch  a  most  active 
warfare  is  waged  between  the  streptococci  and  the  cells. 
Near  the  centre  of  the  patch  there  are  many  free  strep- 
tococci and  a  few  cells.  At  the  margin  there  are  free 
streptococci,  and  also  a  great  many  streptococci  en- 
closed in  cells  (leucocytes)  which  are,  for  the  most  part, 
dead.  In  the  newly-invaded  tissue  we  find  hosts  of 
active  living  cells  engaged  in  eating  up  the  enemies 
as  fast  as  they  can.  The  phagocytologists  tell  us  that  at 
the  centre  the  bacteria  are  fortified,  actively  growing,  and 
virulent  ;  in  the  next  zone  the  leucocytes  which  have 
feasted  upon  the  bacteria  are  poisoned  by  them  ;  outside, 
the  cells,  which  are  more  powerful  and  which  are  con- 
stantly being  reinforced,  are  waging  successful  warfare 
against  the  streptococci.  In  this  manner  the  battle  con- 
tinues, the  cells  now  being  obliged  to  yield  to  the  bacteria 
and  the  patch  spreading,  while  the  cells  subsequently  re- 
inforce and  destroy  the  bacteria,  so  that  the  disease  comes 
to  a  termination. 

Metschnikoff  introduced  fragments  of  tissue  from  ani- 
mals dead  of  anthrax  under  the  skin  of  the  back  of  a  frog, 
and  found  it  surrounded  and  penetrated  by  leucocytes  con- 
taining many  of  the  bacilli. 


IMMUNITY  AND  SUSCEPTIBILITY.  73 

It  need  scarcely  be  pointed  out  that  a  loophole  of  doubt 
exists  in  all  these  illustrations:  the  bacteria  may  have  been 
dead  before  the  cells  ingested  them,  and  the  phenomena  of 
digestion  and  destruction  which  have  gone  on  in  their  in- 
teriors may  have  been  exerted  upon  dead  bacteria.  To  the 
relapsing-fever  illustration  we  may  take  exceptions,  and 
state  that  the  apyrexia  may  have  marked  the  death  of 
the  spirilla,  which  were  taken  up  by  the  leucocytes  only 
when  dead.  In  the  erysipelas  illustration  the  streptococci 
remote  from  the  centre  of  the  lesion  may  have  met  from 
the  body-juices  or  some  other  cause  a  more  speedy  death 
than  that  from  the  digestive  juices  of  the  leucocyte. 

Metschnikoif,  however,  is  prepared  to  show  us  that  the 
leucocytes  do  take  up  living  pathogenic  organisms.  He 
succeeded  in  isolating  two  leucocytes,  each  containing  an 
anthrax  spore,  and  conveying  them  to  artificial  culture- 
media,  where  he  watched  them.  The  new  environment 
being  better  adapted  to  the  growth  of  the  spore  than  for 
the  nourishment  of  the  leucocyte,  the  latter  died,  and 
the  spore  developed  under  his  eyes  into  a  healthy  bacillus. 
Seeing  that  the  animal  cells  take  up  bacteria,  and  seeing 
that  the  ameba  can  ingest  and  digest  "threads  of  lepto- 
thrix  ten  times  as  long  as  itself,"  we  need  only  put  two 
and  two  together  to  see  that  Metschnikoff's  theory  rests 
upon  a  very  substantial  foundation.  The  more  virulent 
the  bacteria,  the  less  ready  the  leucocytes  are  to  seize 
them.  The  more  immune  the  animal,  the  greater  is  the 
affinity  of  the  leucocyte  for  the  bacteria. 

The  organisms  which  are  seized  upon  by  the  leucocytes 
do  not  remain  in  the  blood,  but  are  collected  in  the  spleen 
and  the  lymphatic  glands;  and  not  the  least  important 
.fact  in  favor  of  phagocytosis  is  that  observed  by  Bardach, 
that  excision  of  the  spleen  diminishes  the  resistance  to 
infectious  disease. 

Quinin  also  furnishes  a  therapeutic  support  to  the 
theory.  It  is  known  that  quinin  increases  the  destruc- 
tion of  leucocytes.  Woodhead  inoculated  a  number  of 
rabbits  with  anthrax,  giving  quinin  to  some  of  them. 


74  PATHOGENIC  BACTERIA. 

Those  which  had  received  the  drug  died  earliest— a 
result  probably  dependent  upon  the  destruction  of  part 
of  the  phagocytic  army. 

Ruffer  found  that  the  "phagocytes  evince  a  distinct 
selective  tendency  between  various  kinds  of  organisms. 
They  will  leave  the  bacillus  of  tetanus  in  order  to  seize 
upon  the  Bacillus  prodigiosus  if  simultaneously  intro- 
duced ;  also  the  streptococci  in  diphtheria  for  the  Klebs- 
Loffler  bacilli.  This  is  illustrated  in  the  diphtheritic 
membrane,  where  at  the  surface  one  can  see  leucocytes 
taking  in  numbers  of  the  bacilli,  but  leaving  the  strepto- 
cocci almost  untouched,  with  the  immediate  result  that 
streptococci  are  often  found  in  the  deeper  parts  of  the 
membrane,  and  with  the  remote  result  that  secondary 
abscesses  occurring  in  the  course  of  diphtheria  are  never 
due  to  the  bacillus  of  diphtheria,  but  to  some  other  or- 
ganism." 

Hankin  and  Hardy  found  that  the  three  varieties  of 
leucocytes  in  the  frog's  blood  play  important  parts  in  the 
destruction  of  anthrax  bacilli,  this  destructive  process 
being  accomplished  thus : 

1.  The  eosinophile  cells  are  first  to  approach  and  swal- 
low the  bacteria.      As  this  takes  place  the  eosinophile 
granules  are  seen  to  dissolve  and  act  upon  the  bacteria. 

2.  The  hyaline  cells  take  up  the  remains  of  the  bac- 
teria destroyed  by  the  eosinophile  leucocytes. 

3.  The  basophile  cells  come  to  the  field  loaded  with 
basophilic    granules,    supposed    to   be   antidotal    to   the 
poisons  of  the  bacteria,  surround  the  combatants,  neu- 
tralize the  bacterial  poisons,  and  liberate  the  contesting 
cells. 

Wyssokowitsch  found  that  saprophytic  micro-organ. 
i>ms  are  quickly  eliminated  from  the  blood  when  in- 
jected into  the  circulation.  This  elimination  is  not 
by  excretion  through  organs  nor  by  destruction  in  the 
streaming  blood,  but  by  collection  in  the  small  capil- 
laries, where  the  blood-stream  is  slow  and  where  the 
micro-organisms  are  taken  up  by  the  endothelial  cells. 


IMMUNITY  AND  SUSCEPTIBILITY.  75 

Wyssokowitsch  found  them  most  numerous  in  the  liver, 
spleen,  and  bone-marrow,  and  found  that  in  these  situa- 
tions they  were  destroyed  in  a  short  time — saprophytic 
in  a  few  hours,  pathogenic  in  from  twenty-four  to  forty- 
eight  hours.  Spores  of  Bacillus  subtilis  remained  as 
living  entities  in  the  spleen  for  three  months. 

An  interesting  communication  upon  phagocytosis  is 
that  of  Bordet,  whose  experiments  seem  to  show  that  the 
lack  of  disposition  to  take  up  bacteria  on  the  part  of  the 
leucocytes  may  depend  upon  negative  chemotaxis.  He 
found  that  when  a  guinea-pig  became  very  ill  after  the 
intraperitoneal  introduction  of  a  streptococcus  of  mild 
virulence,  if  an  injection  of  a  culture  of  Proteus  vulgaris 
was  given,  the  leucocytes,  which  had  steadily  refused  to 
take  up  the  streptococci,  seized  upon  the  bacilli  with 
avidity.  This  seems  to  show  that  a  chemical,  or  other 
negative,  or  inhibitory  influence  felt  by  the  leucocyte, 
prevents  it  from  taking  up  all  the  bacteria  that  come 
within  reach. 

4.  THE  HUMORAL  THEORY. — It  was  observed  that  if 
anthrax  bacilli  were  introduced  into  a  few  drops  of 
rabbit's  blood,  they  were  instantly  killed.  This  obser- 
vation was  one  of  immense  importance,  and  from  it  and 
similar  observations  Buchner  deduced  the  principles  of 
his  theory,  which  teaches  that  the  destruction  of  patho- 
genic bacteria  in  the  body  is  due  to  the  bactericidal 
action  of  the  blood-plasma,  not  to  phagocytosis,  which 
phenomenon  amounts  to  nothing  more  than  the  burial 
of  the  dead  bacteria  in  "  cellular  charnel-houses."  The 
experiments  of  Buchner  and  his  followers,  conspicuous 
among  whom  is  Nuttall,  have  shown  that  freshly  drawn 
blood,  blood-plasma,  defibrinated  blood,  aqueous  humor, 
tears,  milk,  urine,  and  saliva  possess  marked  destructive 
influence  upon  the  organisms  brought  in  contact  with 
them — an  influence  easily  destroyed  by  heat. 

The  apparent  paradox  of  rapid  multiplication  of  an- 
thrax bacilli  in  the  rabbit's  blood  enclosed  in  the  rabbit's 
body,  and  the  reversed  action  in  the  test-tube,  caused  im- 


76  PATHOGENIC  BACTERIA. 

mediate  and  prolonged  opposition  to  the  theory.  Each  side 
of  the  question  seemed  well  supported.  The  phagocytolo- 
gists,  however,  showed  that  bacteria  were  often  injured 
and  their  vegetative  powers  destroyed  by  sudden  changes 
from  one  culture-medium  to  another,  this  being  proved 
by  Haffkine,  who  in  experimenting  with  aqueous  humor 
has  shown  that  its  germicidal  actions  are  largely  imagin- 
ary, and  due  to  the  dispersion  of  the  organisms  in  a  large 
amount  of  watery  liquid.  When  the  micro-organisms 
are  introduced  into  it  in  such  a  manner  as  to  remain 
together,  they  grow  well.  If  the  tube  be  shaken,  so  as 
to  distribute  them,  they  die.  Again,  Adami  has  shown 
that  when  blood  is  shed  there  is  almost  always  a  pro- 
nounced destruction  of  corpuscles,  and  suggests  that  the 
antibiotic  property  of  the  shed  blood  may  be  due  to 
solution  of  the  nucleins  formerly  in  the  substance  of  the 
leucocytes.  Jetter  endeavored  to  prove  the  germicidal 
action  of  the  serum  to  be  due  to  certain  salts  which  it 
contained.  His  experiments,  which  consisted  in  observ- 
ing the  action  of  solutions  of  various  salts  in  mixtures 
of  water,  glycerin,  and  gelatin,  were  justly  condemned 
by  Buchner  on  the  ground  that  such  mixtures,  though 
they  might  contain  constituents  of  blood-serum,  were  far 
from  approximating  the  normal  serum  in  composition. 

Wyssokowitsch,  however,  surely  argued  against  hu- 
moral germicide  when  he  showed  that  the  spores  of  Ba^ 
cillus  subtilis  could  reside  in  the  spleen  for  three  months 
uninjured. 

In  supporting  their  theory  the  humoralists  experimented 
by  placing  beneath  the  skin  micro-organisms  enclosed  in 
little  bags  of  pith,  collodium,  etc.  These  bags  allowed 
the  fluids  of  the  body  free  access  to  the  bacteria,  but 
would  shut  out  the  phagocytes.  By  these  means  Hiippe 
and  Lubarsch  have  repeatedly  seen  the  bacteria  grow 
well,  while  the  attempts  of  Batimgarten  have  failed. 
Such  experiments  are  by  no  means  conclusive,  for  we 
should  remember  that  the  operation  necessary  and  the 
presence  of  the  foreign  body  in  which  the  bacteria  are 


IMMUNITY  AND  SUSCEPTIBILITY.  77 

encased  produce  an  inflammatory  transudate  which  may 
have  properties  very  different  from  those  of  the  normal 
juices. 

How  much  of  the  immunity  which  animals  enjoy  de- 
pends upon  the  antibactericidal  action  of  their  body- 
juices  must  remain  an  open  question.  In  some  cases  the 
germicidal  action  of  the  blood  seems  to  be  unquestion- 
able. Buchner  has  shown  that  the  blood-serum  of  ani- 
mals only  possesses  this  germicidal  power  when  freshly 
drawn,  and  that  exposure  of  the  serum  to  sunlight,  its 
mixture  with  the  serum  from  another  species  of  animal, 
its  mixture  with  distilled  water  or  with  dissolved  cor- 
puscles, and  heating  it  to  55°  C.,  check  the  bactericidal 
power.  Buchner  also  points  out  that  the  bactericidal 
and  globulicidal  actions  of  the  blood  are  simultaneously 
extinguished.  Meltzer  and  Norris1  found  that  lymph 
taken  from  the  thoracic  duct  of  the  dog  possessed  marked 
bactericidal  powers  upon  the  typhoid  bacillus. 

The  experiments  of  Pfeiffer  seem  to  add  additional 
support  to  the  humoral  theory  of  immunity.  He  found 
that  when  guinea-pigs  were  given  experimental  choleraic 
peritonitis,  they  could  be  saved  from  death  from  the  affec- 
tion by  intraperitoneal  injection  of  serum  from  an 
immunized  animal.  He  also  showed  that  when  the  cul- 
ture of  cholera,  or  a  culture  cf  typhoid  bacilli,  was  in- 
jected into  the  peritoneum  of  a  guinea-pig,  the  multipli- 
cation of  the  bacteria  was  rapid.  If,  however,  a  few 
drops  of  the  immunized  serum  were  introduced,  a  marked 
effect  was  observed,  for  the  serum  seemed  to  exert  a 
germicidal  effect  upon  the  bacteria,  and  transform  them 
from  living  entities  into  inanimate  little  granular  masses. 

Hankin  is  of  the  opinion  that  the  germicidal  sub- 
stances of  the  blood-serum  are  derived  from  the  eosinophile 
cells,  and  resides  in  the  matter  forming  the  eosin-granules. 

Lowit,2  in  investigating  the  bactericidal  power  of  the 

1  Journal  of  Experimental  Medicine,  vol.  ii.,  No.  6,  p.  701,  Nov.,  1897. 

2  Bietrage  zur  Pathol.  Anatomic  und  zur  Allgem.  Pathologic,  Bd.  xxil.,  H. 
I,  P-  173- 


78  PA  THOGENIC  BACTERIA. 

blood  in  relation  to  its  leucocytes,  found  that  when  a 
marked  experimental  hypoleucocytosis  was  produced,  the 
bactericidal  power  of  the  blood  was  markedly  diminished. 
The  most  interesting  feature  of  his  work  was  the  discov- 
ery that  bactericidal  matter  could  be  extracted  from 
crushed  leucocytes,  and  that  it  could  be  subjected  to  a 
temperature  of  60°  C.  without  change,  thus  differing 
markedly  from  the  alexins. 

Much  discussion  has  arisen  as  to  exactly  what  the  pro- 
tective substances  are.  Buchner  has  applied  the  term 
alexin  to  the  protective  proteid  substances  found  in  the 
blood  of  naturally  immune  animals.  Hankiu  has  given 
us,  together  with  an  extension  of  Buchner' s  idea,  a  con- 
siderable nomenclature  of  somewhat  questionable  utility. 
He  divides  the  protective  substances  (alexins)  into  sozins, 
which  occur  in  the  blood  of  animals  with  natural  immu- 
nity, and  phylaxins,  which  occur  in  the  blood  of  animals 
with  acquired  immunity.  Both  sozins  and  phylaxins  are 
divisible  into  two  groups — thus:  a  sozin  which  acts  de- 
structively upon  bacteria  is  called  a  myco-sozin;  one 
which  neutralizes  bacterial  poisons,  a  toxo-sozin.  A  phy- 
laxin  which  acts  destructively  upon  bacteria  is  called  a 
myco-phylaxin ;  one  which  neutralizes  bacterial  toxins, 
a  toxo-phylaxui. 

The  anti-microbic  serums  obtained  by  PfeifFer,  Kolle, 
Loffler,  and  Abel  from  dogs  and  other  animals  immunized 
to  typhoid  fever  belong  in  the  group  of  myco-phylaxius. 
The  toxo-phylaxins  are  the  antitoxins. 

5.  THE  THEORY  OF  ANTITOXINS.— It  is  a  well-known 
fact  that  individuals  can  accustom  themselves  to  the  use 
of  certain  poisons,  as  tobacco,  opium,  and  arsenic,  so  as 
to  experience  no  inconvenience  from  what  would  be  poi- 
sonous doses  for  other  individuals.  This  is  purely  a 
matter  of  tolerance,  but  is  of  interest  in  connection  with 
the  observations  which  are  to  follow. 

Ehrlich  has  shown  that  animals  can  tolerate  gradually 
increasing  doses  of  ricin  and  abrin,  provided  that  up  to 
a  certain  point  the  increase  of  dosage  is  very  small. 


IMMUNITY  AND  SUSCEPTIBILITY.  79 

When  this  point  is,  however,  safely  passed,  the  increase 
in  dosage  can  be  very  rapid,  yet  without  signs  of  poison- 
ing, seemingly  because  the  drug  is  no  longer  simply  tol- 
erated, but  tolerated  and  simultaneously  neutralized.  By 
experimentation  Ehrlich  has  shown  that  during  the 
period  of  simple  tolerance  the  blood  of  the  animal  is 
unaltered,  but  that  as  soon  as  the  tolerance  becomes 
unlimited  the  blood  contains  a  new  substance,  capable 
not  only  of  protecting  the  animal  by  which  it  is  pro- 
duced, but  also  other  animals  into  whose  blood  it  is  in- 
troduced. In  the  ricin  experiments  this  substance  was 
described  as  antiricin  ;  in  the  experiments  with  abrin,  as 
antiabrin. 

These  investigations  of  Ehrlich  with  the  poisons  of 
higher  plants  succeeded,  but  threw  much  light  upon,  the 
extraordinary  work  of  Behring,  Wernicke,  and  Kitasato, 
who  experimented  with  the  toxins  of  diphtheria  and 
tetanus,  and  showed  that  in  the  blood  of  animals  accus- 
tomed to  these  poisons,  new  substances — antitoxins,  found 
by  Brieger  to  be  proteid  in  nature — were  produced. 

The  antitoxic  theory  of  immunity,  being,  in  the  cases 
cited  at  least,  a  fact  capable  of  demonstration,  has  estab- 
lished itself  at  present  as  the  most  important  hypothesis. 
According  to  it,  acquired  immunity,  at  least,  depends  upon 
the  development  in  the  blood  of  a  neutralizing  substance 
probably  related  to  the  nucleins. 

It  is  of  prime  importance  to  remember  that  the  anti- 
toxin is  an  entirely  new  substance  which  does  not  occur 
in  the  blood  of  normal  animals,  even  when  they  possess 
a  high  degree  of  natural  immunity,  except  in  rare  in- 
stances, and  then  only  in  minute  amounts  not  propor- 
tional to  the  degree  of  immunity.  Calmette  has  called 
special  attention  to  this  fact,  and  points  out  that  while 
fowls  and  tortoises  resist  abrin,  their  blood  contains  no 
anti-abrin;  Vaillard  has  shown  that,  although  the  fowl 
resists  tetanus,  its  blood  contains  no  protective  substance 
destructive  to  tetanus-toxin.  Calmette  finds  that  the 
blood  of  the  ichneumon  and  hedgehog,  which  are  im- 


8o  PA  THOGENIC  BA  CTERIA. 

m  line  to  serpent's  venom,  contains  some  normal  antitoxin, 
but  only  in  small  amount.1  Fischl  and  v.  Wunschheim 
found  a  small  amount  of  a  protecting  substance  in  the 
blood  of  newborn  infants,  which  prevented  the  opera- 
tion of  a  fatal  dose  of  diphtheria  toxin  upon  guinea- 
pigs.2 

Bolton 3  and  the  author  have  found  some  antitoxicity  to 
diphtheria  present  in  the  blood  of  normal  (not  experi- 
mentally immunized)  horses. 

The  origin  of  the  antitoxin  is  a  very  important  and 
interesting  question.  Is  it  in  the  blood,  or  in  all  the 
body  juices?  Does  it  come  from  the  leucocytes?  Dzerj- 
gowsky4  has  estimated  the  quantity  of  antitoxin  con- 
tained in  the  blood  and  organs  of  horses  immunized 
against  diphtheria.  Of  the  constituents  of  the  blood  he 
found  (i)  the  fibrin  has  no  antitoxic  power;  (2)  serum 
obtained  normally  and  that  got  by  expression  from  the 
clot,  from  the  plasma  of  the  same  blood,  have  an  equal 
antitoxic  power;  (3)  the  clot  from  the  plasma,  therefore, 
does  not  retain  the  active  principle;  (4)  the  plasma  and 
the  serum  have  an  equal  antitoxic  power;  (5)  the  red  cor- 
puscles, compared  with  the  plasma,  contain  traces  only 
of  antitoxin;  (6)  serum  containing  the  juice  of  the  leuco- 
cytes is  less  rich  in  antitoxin  than  the  plasma;  (7)  the 
extract  of  the  leucocytes  contains  relatively  little  anti- 
toxin, and  the  leucocytes  themselves  traces  or  none  at  all. 
Hence  the  white  blood-corpuscles  cannot  be  the  place 
where  the  antitoxin  is  formed.  The  serous  liquids  con- 
tained in  organs,  such  as  the  Graafian  follicles,  etc.,  con- 
tain as  much  antitoxin  as  the  blood-serum — none  of  the 
organs  contain  as  much  of  the  antitoxin  as  the  blood 

•If. 

Dzerjgowsky  is  of  the  opinion,   held  probably  by  a 

1  Ann.  de  f  Inst.  Pasteur,  x.,  12. 

.'M-hriftfiir  Hfilkunde,  1895,  xvi.,  429-482. 
1  your,  of  Experimental  Medicine,  vol.  i.,  No.  3,  July,  1896. 
4  Archives  des  Sci.  Biolog.  de  I'/nstitut.  Imper.  de  Med.  Exper.  d  St.  Peters- 
bourg,  tome  V.,  Nos.  2  and  3,  1897. 


IMMUNITY  AND  SUSCEPTIBILITY.  81 

minority  of  scientists,  that  the  antitoxin  is  the  toxin  in 
a  modified  (oxidized  ?)  form,  and  supports  his  view  by 
the  fact  that  the  antitoxins  are  specific  for  their  respec- 
tive toxins  only,  and  by  quoting  the  experiments  of 
Kondrevitsky,  who,  killing  animals  two  hours  after 
an  injection  of  toxin,  found  in  the  blood  toxin  alone ; 
killing  later,  found  some  antitoxin,  and  still  later  much 
antitoxin. 

The  difference  between  this  theory  of  neutralization 
by  antitoxins  and  Chaveau's  retention-hypothesis  is  quite 
marked.  The  retention-theory  teaches  that  a  bacterium 
leaves  behind  it  a  substance  prejudicial  to  its  future 
growth  in  the  economy — a  distinct  metabolic  product. 
The  antitoxic  theory  shows  the  protective  substance  to 
be  a  product  not  of  bacterial  growth,  but  of  tissue-energy, 
not  depending  upon  the  presence  of  the  bacteria,  but 
upon  the  presence  of  a  poison. 

The  antitoxins  do  not  usually  act  harmfully  upon  the 
bacteria,  or  preclude  their  growth  in  the  animal  body,  but 
prevent  their  pathogenesis  by  annulling  their  toxicity— 
i.  e.,  enabling  the  body-cells  to  endure  the  injury — and 
placing  them  in  a  position  exactly  parallel  with  non- 
pathogenic  bacteria. 

Closely  related  to  the  antitoxins,  if  not  identical  with 
them,  are  certain  substances  of  an  anti-infections  nature 
that  can  be  generated  in  the  blood  of  animals  to  which, 
in  the  process  of  immunization,  the  bacteria,  instead  of 
their  poisons,  have  been  administered.  The  anti-infec- 
tious serums  are  protective  against  the  bacterial  infections, 
but  powerless  against  the  toxins.  They  are  the  only 
results  of  immunization  against  cholera  and  typhoid 
fever.  When  antitoxic  serums  can  be  secured  they  are 
of  far  greater  importance,  and  should  always  be  selected 
for  purposes  of  therapeutics. 

The  diseases  which  are  at  present  controllable  by  anti- 
toxins are  toxic  diseases,  caused  by  the  entrance  of  toxin- 
producing  bacteria  into  the  body.  The  growth  of  these 
toxin-producers  probably  depends  upon  the  inability  of 


82  PATHOGENIC  BACTERIA. 

the  body-cells  or  bactericidal  body-juices  to  properly  cope 
with  them,  so  that  they  develop  and  engender  the  poison- 
ous substances  which  are  the  essential  factors  of  disease- 
production.  The  more  the  body  and  its  component  ele- 
ments are  injured,  the  more  successful  the  inroads  of  the 
bacteria,  the  more  prolific  the  toxin-production,  and  the 
more  severe  the  affection. 

The  presence  of  the  antitoxin  annuls  the  poison,  main- 
tains the  vitality  of  the  organism  as  a  whole,  sustains 
the  integrity  of  its  tissues,  and  so  places  the  pathogenic 
bacterium  on  a  very  different  footing  in  relation  to  the 
organism. 

An  antitoxin  is  a  neutralizing  or  annulling  agent,  not 
a  regenerating  one,  and  therefore  in  therapeutics  finds 
its  proper  sphere  only  in  the  beginning  of  the  disease 
combated.  Up  to  a  certain  point  the  symptoms  of  diph- 
theria and  tetanus  are  due  to  the  circulation  of  toxins  in 
the  blood,  and  can  be  successfully  combated  by  antitoxic 
neutralization.  Later  in  both  diseases  we  have  symp- 
toms resulting  from  disorganization  of  the  nervous  sys- 
tem, degeneration  of  the  heart-muscle,  destruction  of  the 
kidneys,  etc.,  and  the  neutralization  of  the  poison  can  be 
of  no  avail  because  the  lesions  are  irreparable,  and  the 
patient  must  succumb. 

I  have  used  the  term  lc  neutralization,"  in  speaking  of 
the  antitoxins,  in  a  rather  free  and  scarcely  warranted 
manner,  and  must  call  attention  to  the  fact  that  their 
operation  is  probably  not  exactly  analogous  to  chemical 
neutralization.  From  mixtures  of  toxin  and  antitoxin 
the  unchanged  poison  has  been  recovered.  The  effect  of 
an  antitoxin  may  be  a  biologic  one,  by  which  the  tissues 
are  so  stimulated  as  to  endue  the  action  of  a  substance 
ordinarily  disorganizing  in  effect. 

Ruchncr  and  Roux  have  both  pointed  out  that  when  the 
t<>xins  and  antitoxins  are  mixed  and  introduced  into  ani- 
mals of  greater  susceptibility  than  are  ordinarily  used,  the 
presence  of  an  unaltered  toxin  can  easily  be  demonstrated. 

This  proof  is,  however,  of  very  little  value,  for  let  the 


IMMUNITY  AND  SUSCEPTIBILITY.  83 

amount  of  toxin  endurance  of  a  resistent  animal  be  repre- 
sented by  x,  and  any  addition  to  this  as  y.  Then  xy 
would  certainly  be  fatal.  If  the  least  quantity  of  anti- 
toxin that  will  protect  the  animal  be  expressed  by  2,  then 
xy  +  z  is  harmless.  It  is  evident,  however,  that  z  does 
not  necessarily  have  any  influence  upon  x,  but  only  need 
neutralize  y  in  order  to  save  the  animal,  and  therefore 
it  is  obvious  that  the  remaining  x  in  such  a  mixture 
could  readily  destroy  another  more  susceptible  animal 
into  which  it  might  be  injected. 

I  am  of  the  opinion  that  the  effect  of  the  antitoxin 
really  partakes  of  the  nature  of  chemic  neutralization 
from  the  following  experiment:  let  x  represent  the  least 
certainly  fatal  dose  of  diphtheria  toxin  for  a  guinea-pig, 
and  y  the  least  quantity  of  antitoxin  that  will  protect 
against  it;  then 

x  -f-  y  is  harmless.     That 

10  x  +  10  y  is  also  harmless  is  known  to  every  one 
accustomed  to  test  antitoxins.  I  have  con- 
tinued this  and  have  found  that 

50  x  -f  50  y 
100  x  +  100  y  are  also  harmless. 

According  to  Buchner,  the  antitoxins  differ  from  the 
alexins  in  being  new  substances  in  the  blood,  in  being 
without  germicidal  or  chemical  neutralizing  power  against 
the  toxins,  and  in  being  stable  compounds  which  can 
resist  heat  to  75°  C.,  can  resist  a  reasonable  amount  of 
exposure  to  light,  and  which  are  not  altered  by  decompo- 
sition of  the  substances  containing  them. 

The  antitoxins  are  specific  for  one  poison  only.  Ehrlich 
found  that  antiricin  was  powerless  against  abrin,  and  vice 
versa.  Diphtheria  antitoxin  is  of  no  avail  against  tetanus, 
and  vice  versa. 

The  immunity  which  the  antitoxins  produce  is  fuga- 
cious, varying  considerably  according,  to  the  particular 
substance  employed.  As  a  rule,  it  is  limited  to  a  few 
months — at  least  in  the  case  of  such  antitoxins  as  we  can 
produce  experimentally. 


84  PA  THOGENIC  BA  CTERIA . 

A  new  principle  discovered  by  Pfeiffer,  and  bearing 
directly  upon  the  theories  of  immunity,  is  that  the  se- 
rum of  animals  immunized  to  certain  diseases  (cholera 
and  typhoid)  contains  a  germicidal  substance.  Metchni- 
koff  has  tried  to  show  that  the  action  of  this  body  depends 
upon  solution  of  the  leucocytes,  but  Pfeiffer  has  disproved 
this  by  showing  that  the  liquor  puris  from  abscesses  oc- 
curring in  the  experiment-animals  did  not  contain  the 
active  substance.1 

The  work  of  van  de  Velde 2  is  very  interesting.  An 
animal  immunized  by  progressively  increasing  doses  of 
strong  filtered  toxin  produced  a  serum  possessed  of  pow- 
erful anti-infectious  and  antitoxic  powers;  one  immu- 
nized by  the  introduction  into  its  body  of  the  washed, 
precipitated  bodies  of  diphtheria  bacilli  collected  by  fil- 
tration furnished  a  serum  of  appreciable  anti-infectious, 
but  no  antitoxic  properties;  one  immunized  by  the  use 
of  bacillus  cultures  developed  antitoxic  and  anti-infectious 
serum  identical  with  the  first  described;  one  immunized 
to  weak  toxin  furnished  serum  of  considerable  anti-infec- 
tious, but  slight  antitoxic  power,  and  still  another  ani- 
mal that  received  toxin  that  had  been  heated  developed 
neither  anti-infectious  nor  antitoxic  serum. 

Seeing  that  the  serums  commercially  manufactured  are 
made  by  the  use  of  strong  filtered  toxin,  van  de  Velde 
examined  a  number  of  samples  purchased  in  the  market, 
and  found  that  they  were  all  possessed  of  both  antitoxic 
and  anti-infectious  properties.  It  is  important  to  remem- 
ber the  presence  of  both  of  these  properties  in  the  serum, 
as  the  successful  use  of  the  agent  for  immunizing  depends 
upon  the  presence  of  the  one,  and  the  use  in  treatment 
upon  the  presence  of  the  other. 

Immunity  and  antitoxins  stand  in  unknown  relation- 
ship to  one  another.  That  an  animal  has  considerable 
antitoxin  in  its  blood  is  no  guarantee  that  it  is  immune. 
I  have  seen  a  horse  in  each  c.cm.  of  whose  blood  there 

1  Centra  ffil.f.  Rakt.  it.  ranisitsnk.,  lid.  xix.,  Nos.  14  and  15. 
*  //'it/.,  Nov.  24,  1897,  Bd.  xxii.,  Nos.  18  and  19. 


IMMUNITY  AND  SUSCEPTIBILITY.  85 

were  300  immunizing  units  of  diphtheria  antitoxin,  die 
of  typical  symptoms  of  diphtheria-poisoning  after  the  ad- 
ministration of  a  comparatively  small  dose  of  the  toxin. 
From  all  that  has  gone  before  it  must  be  clear  to  the 
reader  that  no  single  theory  thus  far  advanced  can  ex- 
plain immunity.  Acquired  immunity  may  depend  in 
the  great  majority  of  cases  upon  antitoxins,  but  as  yet 
we  have  no  satisfactory  explanation  of  natural  immunity. 
The  humoral  theory  may  be  applicable  in  some  cases  ;  in 
others  one  cannot  deny  the  importance  of  the  role  played 
by  the  phagocytes. 


CHAPTER    IV. 
METHODS  OF  OBSERVING  BACTERIA. 

WHOEVER  would  study  bacteria  must  be  equipped  with 
a  good  microscope.  The  instruments  generally  provided 
for  the  use  of  medical  students  in  college  laboratories,  as 
well  as  those  seldom-employed  "show  microscopes  "  seen 
in  physicians'  offices,  are  ill  adapted  for  the  purpose. 
The  essential  features  of  a  bacteriological  instrument 
are  lenses  giving  a  clear  magnification  extending  as 
high  as  one  thousand  diameters,  and  a  good  condenser 
for  intensifying  the  lights  thrown  upon  the  objects.  It 
naturally  follows  that  the  best  work  requires  the  best 
lenses.  The  cheapest  good  microscope  which  is  at  pres- 
ent offered  to  the  public  is  the  BB.  Continental  stand, 
made  by  Bausch  and  Lomb.  This  stand  is  provided  with 
everything  necessary,  is  fitted  with  very  creditable  objec- 
tives, including  an  excellent  -j^"  oil-immersion  lens,  and 
seems  capable  of  doing  very  good  work.  I  do  not 
recommend  this  as  the  best  instrument  obtainable,  but 
as  one  that  is  both  good  and  cheap.  For  those  who  desire 
the  very  best  the  rather  costly  outfits  made  by  Carl  Zeiss 
of  Jena  are  unexcelled. 

For  those  who  may  begin  the  use  of  the  Abbe  con- 
denser and  oil-immersion  lenses  without  the  advantage 
of  personal  instruction  a  few  hints  will  not  be  out  of 
place : 

Always  employ  good  slides  without  bubbles,  and  thin 
cover-glasses ;  No.  i  are  best. 

Place  a  drop  of  oil  of  cedar  upon  the  cover-glass  of 
tlu  sjK-ciim-n  to  be  examined  ;  rack  the  body  of  the  instru- 
ment down  until  the  oil-immersion  lens  touches  the  oil: 

86 


METHODS  OF  OBSERVING  BACTERIA.          87 

keep  on  until  it  almost  touches  the  glass,  then  look  into 
the  microscope  and  find  the  object  by  slowly  and  firmly 
racking  up.  As  soon  as  the  object  comes  into  view 
leave  the  rack  and  pinion  and  focus  with  the  fine  adjust- 
ment. 

Always  select  the  light  from  a  white  cloud  if  possible  ; 
if  there  are  no  white  clouds,  choose  the  clearest  whitest 
light  possible.  Never  under  any  circumstances  employ 
sunlight,  which  is  ruinous  to  the  eyes  and  useful  only 
for  photomicrography. 

In  using  low-power  lenses  the  Abbe  condenser  must  be 
moved  away  from  the  object  and  the  light  modified  by 
the  iris-diaphragm.  The  distance  between  condenser  and 
object  should  correspond  more  or  less  closely  with  the 
distance  between  objective  and  object. 

In  using  high  powers  the  Abbe  condenser  must  be 
brought  near  the  object  and  the  light  modified  by  the 
iris-diaphragm. 

If  the  oil-immersion  lens  is  used,  it  is  perhaps  best  to 
employ  the  plane  side  of  the  mirror.  When  with  this 
lens  a  section  of  tissue  is  examined  for  details,  the  light 
must  be  modified  by  the  iris-diaphragm,  opening  and 
closing  it  alternately  until  the  best  effect  of  illumina- 
tion is  achieved.  If  tissue  be  searched  for  stained  bac- 
teria, and  no  cellular  detail  is  required,  the  diaphragm 
should  be  wide  open  to  admit  a  great  flood  of  light 
and  extinguish  everything  except  the  deeply-colored 
bacteria. 

When  unstained  bacteria  are  to  be  examined  with  the 
oil-immersion  lens,  the  diaphragm  should  be  closed  so 
as  to  leave  only  a  small  opening  through  which  the 
light  can  pass. 

Bacteria  may  be  examined  either  stained  or  unstained. 
The  former  condition  would  always  be  preferable  if  the 
process  of  coloring  the  organisms  did  not  injure  them. 
Unfortunately,  it  is  generally  the  case  that  the  drying, 
heating,  boiling,  macerating,  and  acidulating  to  which 
we  expose  the  organisms  in  the  process  of  staining  alter 


88 


PATHOGENIC  BACTERIA. 


their  shape,  make  their  outlines  less  distinct,  break  up 
their  arrangement,  and  disturb  them  in  a  variety  of  other 
ways.  Because  of  the  possible  errors  of  appearance  re- 
sulting from  these  causes,  as  well  as  because  it  must  be 
determined  whether  or  not  the  individual  is  motile,  in 
making  a  careful  study  of  a  bacterium  it  must  always  be 
examined  in  the  living,  unstained  condition. 

The  simplest  method  of  making  such  an  examination 
would  be  to  take  a  drop  of  the  liquid,  place  it  upon  a 
slide,  put  on  a  cover,  and  examine. 

While  this  method  is  simple,  it  cannot  be  recommended, 
for  if  the  specimen  should  need  to  be  kept  for  a  time 
much  evaporation  takes  place  at  the  edges  of  the  cover- 
glass,  and  in  the  course  of  an  hour  or  two  has  changed  it 
too  much  for  further  use.  The  immediate  occurrence  of 
evaporation  at  the  edges  also  causes  currents  of  liquid  to 
flow  to  and  fro  beneath  the  cover,  carrying  the  bacteria 
with  them  and  making  it  almost  impossible  to  determine 
whether  the  organisms  under  examination  are  motile  or 
not. 

The  best  way  to  examine  living  micro-organisms  is  in 
what  is  called  the  hanging  drop  (Fig.  6).  A  hollow- 


FIG.  6.— The  "hanging  drop"  seen  from  above  and  in  profile. 


ground  slide  is  used,  and  with  the  aid  of  a  small  camel's- 
hair  pencil  a  ring  of  vaselin  is  drawn  on  the  slide  about, 
not  in,  the  concavity  at  its  centre.  A  drop  of  the  mate- 
rial to  be  examined  is  placed  in  the  centre  of  a  large 
clean  cover-glass,  and  then  placed  upon  the  slide  so 


METHODS  OF  OBSERVING  BACTERIA. 


89 


that  the  drop  hangs  in,  but  does  not  touch,  the  concavity. 
The  micro-organisms  are  now  hermetically  sealed  in  an 
air-chamber,  and  appear  under  almost  the  same  con- 
ditions as  in  the  cul- 
ture. Such  a  speci- 
men may  be  kept 
from  day  to  day  and 
examined,  the  bac- 
teria continuing  to 
live  until  the  oxygen 
or  nutriment  is  ex- 
hausted. By  means 
of  a  special  appara- 
tus (Fig.  7),  in  which 
the  microscope  is 
stood,  the  growing 
bacteria  may  be 
watched  at  any  tem- 
perature, and  very 
exact  observations 
made. 

The  hanging  drop 
should  always  be  ex- 
amined at  the  edge, 
as  the  centre  is  too 
thick. 

In  such  a  specimen 

it    is    possible    to    de-         FIG.  7. — Apparatus  for  keeping  objects  under 
•termilie     the     shape,     microscopic  examination  at  constant   tempera- 

size,  grouping,  divis-   tures< 

ion,    sporulation,   and   motility  of  the   organism   under 

observation. 

Care  should  be  exercised  to  use  a  rather  small  drop, 
especially  for  the  detection  of  motility,  as  a  large  one 
vibrates  very  readily  and  masks  the  motility  of  the 
sluggish  forms. 

When  the  bacteria  to  be  observed  are  in  solid  or  semi- 
solid  culture,  a  small  quantity  of  the  culture  should  be 


90  PATHOGENIC  BACTERIA. 

mixed  up  in  a  drop  of  sterile  bouillon  or  water  and  ex- 
amined. 

In  the  early  days  of  study  efforts  were  made  to  facili- 
tate the  observation  of  bacteria  by  the  use  of  carmin  and 
hematoxylon.  Both  of  these  reagents  tinge  the  proto- 
plasm of  the  organisms  a  little,  but  so  unsatisfactorily 
that  since  Weigert  introduced  the  anilin  dyes  for  the 
purpose  both  of  these  tissue-stains  have  been  rejected. 
The  affinity  between  the  bacteria  and  the  anilin  dyes  is 
peculiar,  and  many  times  is  so  certain  a  reaction  as  to 
become  an  essential  factor  in  the  differentiation  of 
species. 

For  the  study  of  bacteria  in  the  stained  condition  we 
now  employ  the  anilin  dyes  only.  These  wonderful 
colors,  as  numerous  as  the  rainbow  hues,  are  coal-tar 
products.  Huppe  classifies  them  as  follows  : 

A.  Dyes  prepared  from  anilin  oil. 

1.  Oxidation-products  of  pure  anilin  : 

Methylene  blue, 

Chlorhydrin  blue  (basic  indulin). 

2.  Oxidation-products  of  pure  toluol  : 

Safranin. 

3.  Oxidation-products  of  mixed  anilin  and  toluol  : 

(a)  Rosanilin.     When  pure  this  is  triamido- 

diphenyl-toluyl-karbinol. 

Fuchsin — rosanilin  hydrochlorate.  It  is 
often  mixed  with  the  acetate  and  the 
pararosanilin  acetate  and  hydrochlo- 
rate. The  pure  rosanilin  hydrochlorate 
should  always  be  chosen  for  purposes  of 
staining. 

Azalein  is  rosanilin  nitrite. 

Methylized  and  ethylized  rosanilin  : 
lodin  violet, 
Dahlia, 
lodin  green. 

(b)  Pararosanilin.     The  colorless   pure   Fara- 

rosanilin  is  triamido-triphenyl-karbinol. 


METHODS  OF  OBSERVING  BACTERIA.          91 

Rubin-pararosanilin  hydrochlorate. 
Methylized,     ethylized,     and    benzylized 
pararosanilid : 

Crystal  violet, 

Gentian  violet, 

Victoria  blue, 

Methyl  green, 

Auramin. 

The  rosanilins  are  more  difficult  to  prepare 
than  the  pararosanilins,  and  are  generally 
mixed  with  them.  The  pararosanilins 
color  more  sharply  than  the  rosanilins. 

4.  Amido-azo  combinations : 

Bismarck  brown, 
Phenylene  brown, 
Vesuvin. 

5.  Chinolin  derivatives : 

Cyanin. 

B.   Naphthalin  group. — Magdala  red. 

The  best  anilin  dyes  made  at  the  present  time,  and 
those  which  have  become  the  standard  for  all  bacterio- 
logical work,  are  made  in  Germany  by  Dr.  Griibler.  In 
ordering  the  stain  the  name  of  this  manufacturer  should 
always  be  specified. 

A  whole  volume  could  easily  be  devoted  to  scientific 
staining.  Indeed,  the  technical  difficulties  encountered 
are  so  great  that  no  explanations  can  be  too  thorough  to 
be  useful.  The  special  methods  essential  for  such  bac- 
teria as  have  peculiar  staining  reactions  will  be  given 
with  the  description  of  the  organism.  General  methods 
only  will  be  discussed  in  this  chapter. 

Cover-glass  Preparations  for  General  Examination. 
— The  material  to  be  examir.ed  must  be  spread  in  the 
thinnest  possible  layer  upon  the  surface  of  a  perfectly 
clean  cover-glass,  and  dried.  Here  it  may  be  remarked 
that  for  bacteriological  purposes  thin  covers  (No.  i)  are 
generally  required,  because  thick  glasses  interfere  with 
the  focussing  of  the  oil-immersion  lenses,  and  that  cover- 


92  PATHOGENIC  BACTERIA. 

glasses  can  never  be  too  clean.  It  is  best  to  immerse 
them  first  in  a  strong  mineral  acid,  then  to  wash  them  in 
water,  then  in  alcohol,  then  in  ether,  and  keep  them  in 
ether  until  they  are  to  be  used.  Except  that  it  some- 
times cracks,  bends,  or  fuses  the  edges  of  the  glasses,  a 
better  and  more  convenient  method  of  cleaning  them  is  to 
wipe  them  as  clean  as  possible,  seize  them  in  fine-pointed 
forceps,  pass  them  repeatedly  through  a  small  Bnnsen 
flame  until  it  becomes  greenish  yellow,  then  slowly  ele- 
vate the  glasses  above  the  flame,  so  as  to  allow  them  to 
anneal.  This  maneuvre  removes  the  organic  matter  by 
combustion.  It  is  not  expedient  to  use  covers  twice  for 
bacteriological  work,  though  if  well  cleaned  they  may 
subsequently  be  employed  for  ordinary  microscopic  ob- 
jects. 

To  return :  After  the  material  spread  upon  the  cover 
has  dried,  it  must  be  fixed  to  the  glass  by  immersion  for 
twenty-four  hours  in  equal  parts  of  absolute  alcohol  and 
ether,  or,  as  is  much  easier  and  more  rapid,  be  passed 
three  times  through  a  flame.  Three  is  not  a  magic 
number,  but  experience  has  shown  that  when  drawn 
through  the  flame  three  times  the  desired  effect  seems 
best  accomplished.  The  Germans  recommend  that  a 
Bunsen  burner  or  a  large  alcohol  lamp  be  used,  that  the 
arm  holding  the  forceps  containing  the  cover-glass  in- 
scribe a  circle  a  foot  in  diameter,  and  that,  as  each  revo- 
lution occupies  a  second  of  time,  the  glass  be  made  to  pass 
through  the  flame  from  apex  to  base  three  times.  This 
is  supposed  to  be  exactly  the  requisite  amount  of  heating. 
The  rule  is  a  good  one  for  the  inexperienced. 

After  fixing,  the  material  is  ready  for  the  stain.  Every 
laboratory  should  be  provided  with  several  stock-solutions 
of  the  more  ordinary  dyes.  These  stock-solutions  are 
saturated  alcoholic  solutions  made  by  adding  25  grams 
of  the  dye  to  100  c.cm.  of  alcohol.  Of  these  it  is  well  to 
have  fuchsin,  -i-ntian  violet,  and  methylene  blue  always 
made  up.  The  stock -solutions  will  not  stain,  but  are  the 
standards  for  the  manufacture  of  the  working  stains. 


METHODS  OF  OBSERVING  BACTERIA.          93 

For  ordinary  staining  an  aqueous  solution  made  in  a 
simple  manner  is  employed.  A  small  bottle  is  nearly 
rilled  with  distilled  water,  and  the  stock-solution  is  added, 
drop  by  drop,  until  the  color  becomes  just  sufficiently  in- 
tense to  prevent  the  ready  recognition  of  objects  through 
it.  Such  a  watery  solution  possesses  the  power  of  readily 
penetrating  the  dried  protoplasm  of  the  bacterium,  taking 
the  stain  with  it.  Alcohol  does  not  have  this  power. 

As  in  the  process  of  staining  the  cover  is  apt  to  slip 
from  the  fingers  and  spill  the  stain,  it  is  well  to  be  pro- 
vided with  cover-glass  forceps  (Fig.  8),  which  hold  the 


FIG.  8. — Stewart's  cover-glass  forceps. 

glass  in  a  firm  grip  and  allow  of  all  manipulations  with- 
out danger  to  the  fingers  or  clothes.  The  ordinary  in- 
struments are  entirely  unfitted  for  the  purpose,  as  capil- 
lary attraction  draws  the  stain  between  the  blades  and 
makes  certain  the  soiling  of  the  fingers.  Sufficient  stain 
is  allowed  to  run  from  a  pipette  upon  the  smeared  side 
of  the  cover-glass  to  flood  it,  but  not  overflow,  and  is 
allowed  to  remain  for  a  moment  or  two,  after  which  it 
is  thoroughly  washed  off  with  water.  If  the  specimen 
is  one  prepared  for  temporary  use,  it  can  be  examined  at 
once,  mounted  in  a  drop  of  water,  but  under  these  con- 
ditions will  not  appear  as  advantageously  as  if  dried  and 
then  mounted  in  Canada  balsam. 

Sometimes  the  material  to  be  examined  is  too  solid  to 
spread  upon  the  glass  conveniently.  Under  such  circum- 
stances a  drop  of  distilled  water  can  be  added  and  a  minute 
portion  of  the  material  be  mixed  in  it  upon  the  glass. 

The  entire  process  is,  in  brief : 

i.  Spread  the  material  upon  the  cover  ;  2.  Dry — do  not 
heat ;  3.  Pass  three  times  through  the  flame  ;  4.  Stain 


94  PATHOGENIC  BACTERIA. 

two  to  three  minutes;  5.  Wash  thoroughly  in  water; 
6.  Dry;  7.  Mount  in  Canada  balsam. 

This  simple  process  suffices  to  stain  most  bacteria. 

Ohlmacher1  deserves  credit  for  his  observation  that 
when  the  "fixed  "  preparation  is  immersed  for  a  moment 
or  two  in  a  2-4  per  cent,  solution  of  formalin,  the  brill- 
iancy of  the  stain  is  considerably  increased. 

Staining  Bacteria  in  Sections  of  Tissue.  —  It  not 
infrequently  happens  that  the  bacteria  to  be  examined 
are  scattered  among  or  enclosed  in  the  cells  of  tissues. 
Their  demonstration  is  then  a  matter  of  some  difficulty, 
and  the  method  employed  is  one  which  must  be  modified 
according  to  the  kind  of  organism  to  be  stained.  Very 
much,  too,  depends  upon  the  preservation  of  the  tissue 
to  be  studied.  As  bacteria  disintegrate  rapidly  in  dead 
tissue,  the  specimen  for  examination  should  be  secured 
as  fresh  as  possible,  cut  into  small  fragments,  and  im- 
mersed in  absolute  alcohol  from  six  to  twenty-four  hours 
to  kill  the  cells  and  bacteria.  Afterward  they  are  re- 
moved from  the  absolute  alcohol  and  kept  in  80-90 
per  cent.,  which  does  not  shrink  the  tissue.  Bichlorid 
of  mercury  may  also  be  used,  but  absolute  alcohol  seems 
to  answer  every  purpose. 

The  ordinary  methods  of  imbedding  suffice.  The  sim- 
pler of  these  are  probably  as  follows: 

I.  Celloidin. — From  the  hardening  reagent  (if  other 
than  absolute  alcohol) — 

12-24  hours  in  95  per  cent,  alcohol, 

6-12      "      "  absolute  alcohol, 
12-24  '  thin  celloidin  (consistence  of  oil), 

6-12  "  thick  celloidin  (consistence  of  molasses). 

The  solutions  of  celloidin  are  made  in  equal  parts  of 
absolute  alcohol  and  ether. 

Place  upon  a  block  of  dry  wood,  allow  to  evaporate 
until  the  block  can  be  overturned  without  dislodging  the 
spc-cmu-u  ;  then  place  in  70-80  per  cent,  alcohol  until 

Mnm,  l-vi).  1 6,  1896. 


METHODS  OF  OBSERVING  BACTERIA.          95 

ready  to  cut.     The  knife  must  be  kept  flooded  with  alco- 
hol while  cutting. 

II.  Paraffin— 

12-24  hours  in  95  per  cent,  alcohol, 
6-12      "      "  absolute  alcohol, 

4      "      u  chloroform,  benzole,  or  xylol, 
4-8      u      "a  saturated  solution  of  paraffin  in  one  of 
the  above  reagents. 

Place  in  melted  paraffin  in  an  oven  or  paraffin  water- 
bath,  at  4o°-45°  C.,  until  the  volatile  reagent  is  all  evap- 
orated, and  the  tissue  impregnated  with  paraffin.  Im- 
bed in  freshly  melted  paraffin  in  any  convenient  mould. 
In  cutting,  the  knife  must  be  perfectly  dry. 

When  it  is  necessary,  subsequently,  to  remove  the  im- 
bedding material,  dissolve  the  paraffin  in  chloroform, 
benzole,  xylol,  oil  of  turpentine,  etc.,  which  in  turn  can 
be  removed  with  95  per  cent,  alcohol. 

Celloidin  is  soluble  in  absolute  alcohol,  ether,  and  oil  of 
cloves.  It  is  very  convenient  to  fasten  the  cut  sections  upon 
the  slide — paraffin  sections  by  oil  of  cloves  and  collodion 
or  gum  arabic  solution,  celloidin  sections  by  firmly  pressing 
filter  paper  upon  them  and  rubbing  hard,  then  allowing 
a  little  vapor  of  ether  to  run  upon  them. 

III.  Glycerin-Gelatin. — As  the  penetration  of  the  tissue 
by  celloidin  is  attended  with  lessened  staining-qualities  of 
the  tubercle  bacillus,  it  has  been  recommended  by  Kolle1 
that  the  tissue  be  saturated  with  a  mixture  of  glycerin,  i 
part;  gelatin,  2  parts;  and  water,  3  parts;  cemented  to  a 
cork  or  block  of  wood,  hardened  in  absolute  alcohol  and 
cut  as  usual  for  celloidin  with  a  knife  wet  with  alcohol. 

For  staining  bacteria  (other  than  the  tubercle  and 
lepra  bacilli)  in  tissue,  two  universal  methods  can  be 
recommended: 

Loffler's  Method. — The  cut  sections  of  tissue  are 
stained  for  a  few  minutes  in  Loffler's  alkaline  methylene- 
blue  solution  (q.  ^.),  and  then  differentiated  in  a  i  per 

1  Flugge's  Mikroorganismen. 


96  PATHOGENIC  BACTERIA. 

cent,  solution  of  hydrochloric  acid  for  a  few  seconds. 
The  section  is  subsequently  dehydrated  in  alcohol,  cleared 
up  in  xylol,  and  mounted  in  balsam. 

Pfeiffer's  Method. — The  sections  are  stained  for  one- 
half  hour  in  diluted  ZiehPs  carbol-fuch^in  (q.  v.\  then 
transferred  to  absolute  alcohol  made  feebly  acid  with 
acetic  acid.  The  sections  must  be  carefully  watched, 
and  as  soon  as  the  original,  almost  black-red  color  gives 
place  to  a  red  violet  color  the  section  is  removed  to 
xylol,  where  it  is  cleared  preparatory  to  mounting  in 
balsam. 

For  ordinary  work  the  following  simple  method  is 
recommended:  After  the  sections  are  cut  the  paraffin 
must  be,  and  the  celloidin  had  better  be,  removed. 
From  water  the  sections  are  placed  in  the  same  watery 
stain  used  for  cover-glasses  and  allowed  to  remain  five 
to  eight  minutes.  They  are  next  washed  in  water  for 
several  minutes,  then  decolorized  in  0.5-1  per  cent, 
acetic-acid  solution.  The  acid  removes  the  stain  from 
the  tissues,  and  ultimately  from  the  bacteria  as  well, 
so  that  one  must  watch  carefully,  and  as  soon  as  the 
color  almost  disappears  from  the  sections  remove  them 
to  absolute  alcohol.  At  this  point  the  process  may  be 
interrupted  to  allow  the  tissue-elements  to  be  counter- 
stained  with  alum  carmin  or  any  stain  not  requiring 
acid  for  differentiation,  after  which  the  sections  are 
dehydrated  in  absolute  alcohol,  cleared  in  xylol,  and 
mounted  in  Canada  balsam. 

fa  will  be  mentioned  hereafter,  certain  of  the  bacteria 
which  occur  in  tissue  do  not  allow  of  the  ready  penetra- 
tion of  the  color.  For  such  forms  a  more  intense  stain 
must  l>t-  employed.  One  of  the  best  of  these  stains, 
which  can  be  employed  by  the  given  method  both  for 
cover-glasses  and  tissues,  is  Loffler's  alkaline  methylene 
blue  : 

Saturated  alcoholic  solution  of  methylene  blue,  30 ; 
i :  10,000  aqueous  solution  of  caustic  potash,    100. 


METHODS  OF  OBSERVING  BACTERIA.          97 

Some  bacteria,  as  the  typhoid-fever  bacillus,  decolorize 
so  rapidly  as  to  contraindicate  the  use  of  acid  for  the  dif- 
ferentiation, washing  in  water  or  alcohol  being  sufficient. 

Gram's  Method  of  Staining  Bacteria  in  Tissue. — 
Gram  was  the  fortunate  discoverer  of  a  method  of  stain- 
ing bacteria  in  such  a  manner  as  to  saturate  them  with 
an  insoluble  color.  It  will  be  seen  at  a  glance  what  a 
marked  improvement  this  is  on  the  method  given  above, 
for  now  the  stained  tissue  can  be  washed  thoroughly  in 
either  water  or  alcohol  until  its  cells  are  colorless,  with- 
out fear  that  the  bacteria  will  be  decolorized.  Its  prose- 
cution is  as  follows  :  The  section  is  stained  from  five  to 
ten  minutes  in  a  solution  of  a  basic  anilin  dye — pure 
anilin  (anilin  oil)  and  water.  This  solution,  first  devised 
by  Ehrlich,  is  known  as  Ehrlich's  solution.  The  ordinary 
method  of  preparing  it  is  to  mix  the  following : 

Pure  anilin,  4  5 

Saturated  alcoholic  solution  of  gentian  violet,     n  ; 
Water,  100. 

Instead  of  gentian  violet,  methyl  violet,  fuchsin,  or  any 
basic  anilin  color  may  be  used.  The  mixture  does  not 
keep  well— in  fact,  seldom  longer  than  six  to  eight  weeks, 
sometimes  not  more  than  two  or  three  ;  therefore  it  is 
best  to  prepare  it  in  very  small  quantity  by  pouring 
about  i  c.cm.  of  pure  anilin  into  a  test-tube,  filling 
the  tube  about  one-half  with  distilled  water,  shaking 
the  mixture  well,  then  filtering  as  much  as  is  desired 
into  a  small  dish.  To  this  the  saturated  alcoholic  solu- 
tion of  the  basic  dye  is  added  until  the  surface  becomes 
distinctly  metallic  in  appearance. 

Friedlander  recommends  that  the  section  remain  from 
fifteen  to  thirty  minutes  in  warm  stain,  and  in  many  cases 
the  prolonged  process  gives  better  results. 

From  the  stain  the  section  is  given  a  rather  hasty  wash- 
ing in  water,  and  then  immersed  from  two  to  three  min- 
utes in  Grain's  solution  (a  dilute  Lugol's  solution)  : 


98  PATHOGENIC  BACTERIA. 

lodin  crystals,  i  5 

Potassium  iodid,  2  ; 

Water,  3°°- 

While  the  specimen  is  in  the  Gram's  solution  it 
appears  to  turn  a  dark  blackish-brown  color.  When 
removed  from  the  solution  it  is  carefully  washed  in  95 
per  cent,  alcohol  until  no  more  color  is  given  off  and 
the  tissue  assumes  a  grayish  color.  If  it  is  simply 
desired  to  find  the  bacteria,  the  section  is  dehydrated 
in  absolute  alcohol  for  a  moment,  cleared  up  in  xylol, 
and  mounted  in  Canada  balsam.  If  it  is  necessary  to 
study  the  relation  between  the  bacteria  and  the  tissue- 
elements,  a  nuclear  stain,  such  as  alum  carmin  or  Bis- 
marck brown,  may  be  subsequently  used.  Should  a 
nuclear  stain  requiring  acid  for  its  differentiation  be 
desirable,  the  process  of  staining  must  precede  the  Gram 
method  altogether,  so  that  the  acid  shall  not  act  upon 
the  stained  bacteria. 

The  success  of  Gram's  method  rests  upon  the  fact  that 
the  combination  of  mycoprotcin,  basic  anilin,  and  the 
indicts  forms  a  compound  insoluble  in  alcohol. 

The  process  described  may  be  summed  up  as  follows  : 

Stain  in  Ehrlich's  anilin-water  gentian  violet  five 

to  thirty  minutes ; 
Wash  momentarily  in  water ; 
Immerse  two  to  three  minutes  in  Gram's  solution  ; 
Wash  in  95  per  cent,  alcohol  until  no  more  color 

comes  out ; 

Dehydrate  in  absolute  alcohol ; 
Clear  up  in  xylol ; 
Mount  in  Canada  balsam. 

This  method  stains  a  large  variety  of  bacteria  very 
beautifully,  but,  unfortunately,  does  not  stain  them  all, 
and  as  some  of  those  which  do  not  stain  are  important, 
it  seems  well  to  mention  the — 


METHODS  OF  OBSERVING  BACTERIA.          99 

Spirillum  of  cholera  and  of  chicken-cholera ; 
Bacillus  mallei  (of  glanders) ; 
Bacillus  of  malignant  edema  ; 
Bacillus  pneumonise  of  Friedlander ; 
Micrococctis  gonorrhceae  of  Neisser  ; 
Spirochaete  Obermeieri  of  relapsing  fever ; 
Bacillus  of  typhoid  fever  ; 
Bacillus  of  rabbit-septicemia. 

Gram's  method  is  a  method  of  staining  bacteria  in 
tissues,  but  the  fact  that  the  method  colors  some  but  not 
all  bacteria  is  one  of  considerable  importance  from  a  dif- 
ferential point  of  view  ;  and  as  the  difficulty  of  separating 
the  species  of  bacteria  is  so  great  that  every  such  point 
must  be  eagerly  seized  for  assistance,  this  method  be- 
comes one  much  employed  for  cover-glass  preparations, 
where  it  is  more  easily  performed  than  for  sections. 

Gram's  Method  for  Cover-glass  Preparations. — A 
thin  layer  of  the  bacteria  to  be  examined  is  spread  upon 
the  cover-glass,  dried,  and  fixed.  The  cover,  held  in  the 
grip  of  a  cover-glass  forceps,  is  flooded  with  Ehrlich's 
solution.  By  holding  the  cover  flooded  with  stain  over 
a  small  flame  for  a  moment  or  two  the  solution  is  kept 
warm,  and  the  process  of  staining  is  continued  from  two 
to  five  minutes.  If  the  heating  causes  the  stain  to 
evaporate,  more  of  it  must  be  dropped  upon  the  glass, 
so  that  it  does  not  dry  up  and  incrust. 

The  stain  is  poured  off,  and  the  cover  placed  in  a  small 
dish  of  Gram's  solution  and  allowed  to  remain  one-half 
to  two  minutes,  the  solution  being  agitated.  It  is  pos- 
sible to  apply  the  Gram  solution  in  the  same  manner 
in  which  the  stain  is  used,  but  as  a  relatively  larger 
quantity  should  be  employed,  the  dish  seems  preferable. 

The  cover  is  next  washed  in  95  per  cent,  alcohol  until 
the  blue  color  is  wholly  %or  almost  lost,  after  which  it  can 
be  counter-stained  with  eosin,  Bismarck  brown,  vesuvin, 
etc.,  washed,  dried,  and  mounted  in  Canada  balsam. 
Given  briefly,  the  method  is : 


100  PATHOGENIC  BACTERIA. 

Stain  with  Ehrlich's  solution  two  to  five  minutes  ; 
Gram's  solution  for  one-half  to  two  minutes  ; 
Wash  in  95  per  cent,  alcohol  until  decolorized  ; 
Counter-stain  if  desired  ;  wash  the  counter-stain 

off  with  water ; 
Dry; 
Mount  in  Canada  balsam. 

Method  of  Staining  Spores. — It  has  already  been 
remarked  that  the  peculiar  quality  of  the  spore-capsules 
protects  them  from  the  influence  of  stains  and  disinfect- 
ants to  a  certain  extent.  On  this  account  they  are  much 
more  difficult  to  color  than  the  adult  bacteria.  Several 
methods  are  recommended,  the  one  generally  employed 
being  as  follows :  Spread  the  thinnest  possible  layer  of 
material  upon  a  cover-glass,  dry,  and  fix.  Have  ready 
a  watch-crystalful  of  Ehrlich's  solution,  preferably  made 
of  fuchsin,  and  drop  the  cover-glass,  prepared  side  down, 
upon  the  surface,  where  it  should  float.  Heat  the  stain 
until  it  begins  to  steam,  arid  allow  the  specimen  to 
remain  in  the  hot  stain  for  five  to  fifteen  minutes.  The 
cover  is  now  transferred  to  a  3  per  cent,  solution  of  hydro- 
chloric acid  in  absolute  alcohol  for  about  one  minute. 
Abbott  recommends  that  the  cover-glass  be  submerged, 
prepared  side  up,  in  a  dish  of  this  solution  and  gently 
agitated  for  exactly  one  minute,  then  removed,  washed 
in  water,  and  counter-stained  with  an  aqueous  solution 
of  methyl  or  methylene  blue. 

In  such  a  specimen  the  spores  should  appear  red,  the 
bacilli  blue. 

I  have  not  generally  found  that  spores  color  so  easily, 
and  for  many  species  the  best  method  seems  to  be  to 
place  the  prepared  cover-glass  in  a  test-tube  half  full  of 
carbol-fuchsiu  : 

Fuchsin,  T  . 

Alcohol,  IO ; 

5  per  cent,  aqueous  solution  of  phenol  crystals,   100, 


t            r  .  .          ,               ,         ,           ,,,,,.           , 

,  -      •  r     •••*.•*•        »  »V» 

•  '    ' ;' ;      I  •*  '  .'  •„      '•••••»  I  • 

•  '.•'••'  .,',,' ',  '••',*•',,,',  *    ', 


METHODS  OF  OBSERVING  BACTERIA.        101 

and  boil  it  for  at  least  fifteen  minutes,  after  which  it  is 
decolorized,  either  with  3  per  cent,  hydrochloric  or  2-5  per 
cent,  acetic  acid,  washed  in  water,  and  counter-stained  blue. 

Fiocca  suggests  the  following  rapid  method:  "About 
20  c.cm.  of  a  10  per  cent,  solution  of  ammonium  are 
poured  into  a  watch-glass,  and  10-20  drops  of  a  saturated 
solution  of  gentian  violet,  fuchsin,  methyl  blue,  or  saf- 
ranin  added.  The  solution  is  warmed  until  vapor  begins 
to  rise,  then  is  ready  for  use.  A  very  thinly-spread  cover- 
glass,  carefully  dried  and  fixed,  is  immersed  for  three  to 
five  minutes  (sometimes  ten  to  twenty  minutes),  washed 
in  water,  washed  momentarily  in  a  20  per  cent,  solution 
of  nitric  or  sulphuric  acid,  washed  again  in  water,  then 
counter-stained  with  a  watery  solution  of  vesuvin,  chrys- 
oidin,  methyl  blue,  malachite  green,  or  safranin,  according 
to  the  color  of  the  preceding  stain.  This  whole  process 
is  said  to  take  only  from  eight  to  ten  minutes,  and  to  give 
remarkably  clear  and  beautiful  pictures." 

Method  of  Staining  Flagella, — This  is  much  more 
difficult  than  the  staining  of  either  the  bacteria  or  their 
spores,  because  each  species  seems  to  behave  differently 
in  its  relation  to  the  stain,  so  that  the  chemistry  of  the 
micro-organismal  products  must  be  taken  into  considera- 
tion. 

The  best  method  introduced  is  that  of  Loffler.  In  it 
three  solutions  are  used  : 

A.  A  20  per  cent,  solution  of  tannic  acid,  10 ; 
Cold  saturated  aqueous  solution  of  ferrous  sulphate,    5  ; 
Alcoholic  solution  of  fuchsin  or  methyl  violet,  i. 

B.  A  i  per  cent,  solution  of  caustic  soda. 

C.  An  aqueous  solution  of  sulphuric  acid  of  such  strength 

that  i  c.cm.  will  exactly  neutralize  an  equal  quan- 
tity of  Solution  B. 

Some  of  the  bacteria  to  be  stained  are  mixed  upon  a 
cover-glass  with  a  drop  of  distilled  water.  This  is  the 
first  dilution,  but  is  too  rich  in  bacteria  to  allow  the 


102  PATHOGENIC  BACTERIA. 

flagella  to  show  well,  so  that  it  is  recommended  to  prepare 
a  second  dilution  by  placing  a  small  drop  of  distilled 
water  upon  a  cover  and  taking  a  small  portion  from  the 
first  cover  to  the  second,  spreading  it  over  the  entire  sur- 
face. The  material  is  allowed  to  dry,  and  is  then  fixed 
by  passing  it  three  times  through  the  flame.  When  this 
is  done  with  forceps  there  is  some  danger  of  the  prepara- 
tion becoming  too  hot,  so  Loffler  recommends  that  the 
glass  be  held  in  the  fingers  while  the  passes  through  the 
flame  are  made. 

The  cover-glass  is  now  held  in  forceps,  and  the  mordant, 
Solution  A,  is  dropped  upon  it  until  it  is  well  covered. 
The  cover  is  warmed  until  it  begins  to  steam,  and  the 
stain  replaced  as  it  evaporates.  It  must  not  be  heated  too 
strongly  ;  above  all  things,  must  not  boil.  This  solution 
is  allowed  to  act  from  one-half  to  one  minute,  is  then 
washed  in  distilled  water,  then  in  absolute  alcohol  until  all 
traces  of  the  solution  have  been  removed.  The  real  stain 
— Loffler  recommends  an  anilin-water  fuchsin  (Ehrlich's 
solution) — which  should  have  a  neutral  reaction,  is  now 
dropped  on  so  as  to  cover  the  specimen,  and  heated  for  a 
minute  until  vapor  begins  to  arise;  it  is  then  washed  off 
carefully,  dried,  and  mounted  in  Canada  balsam.  To 
obtain  this  neutral  reaction  enough  of  the  i  per  cent, 
sodium-hydrate  solution  is  added  to  an  amount  of  the 
anilin-water-fuchsin  solution  having  a  thickness  of  sev- 
eral centimeters  to  begin  to  change  the  transparent  into 
an  opaque  solution.  Such  a  specimen  may  or  may  not 
show  the  flagella.  If  not,  before  proceeding  farther  it  is 
necessary  to  study  the  products  of  the  bacterium  in  cul- 
ture-media. If  by  its  growth  the  organism  elaborates 
alkalies,  Solution  C,  in  proportion  from  i  drop  to  i  c.cm. 
in  1 6  c.cm.  of  the  mordant  A,  must  be  added,  and  the 
process  repeated  a^ain  and  a^ain  until  the  proper  amount 
is  determined.  On  the  other  hand,  if  the  organism  by 
its  growth  produces  acid,  Solution  B  must  be  added, 
drop  by  drop,  until  i  in  16  cm.  have  been  attained,  and 
numerous  experiments  made  to  see  when  the  flagella 


METHODS  OF  OBSERVING  BACTERIA.        103 

will  appear.  Loffler  has  fortunately  worked  out  the 
amounts  required  for  some  of  the  species,  and  of  the 
more  important  ones  the  following  amounts  of  Solutions 
B  and  C  must  be  added  to  16  c.cm.  of  Solution  A  to 
attain  the  desired  effect : 

Cholera  spirillum,  ^-i  drop  of  Solution  C  ; 

Typhoid  fever,  i  c.cm.  of  Solution  B  ; 

Bacillus  subtilis,  28-30  drops  of  Solution  B  ; 

Bacillus  of  malignant  edema,  36-37  drops  of  Solution  B. 

Part  of  the  success  of  the  staining  depends  upon 
having  the  bacteria  thinly  spread  upon  the  glass,  and  as 
free  from  albuminous  and  gelatinous  materials  as  possi- 
ble. The  cover-glass  must  be  cleaned  most  painstakingly  : 
too  much  heating  in  fixing  must  be  avoided.  After  using 
and  washing  off  the  mordant,  the  preparation  should  be 
dried  before  the  application  of  the  anilin-water-fuchsin 
solution. 

Pitfield l  has  devised  a  simple  and  good  method  of 
staining  flagella.  A  single  solution  at  once  mordant  and 
stain  is  employed.  It  is  made  in  two  parts,  which  are 
filtered  and  mixed. 

A.  Saturated  aqueous  solution  of  alum,  10  c.cm. ; 
Saturated  alcoholic  solution  of  gentian-violet,  i  c.cm. 

B.  Tannic  acid,  i  gr. ; 
Distilled  water,                                                     10  c.cm. 

The  solutions  should  be  made  with  cold  water,  and 
immediately  after  mixing  the  stain  is  ready  for  use.  The 
cover-slip  is  carefully  cleaned,  the  grease  being  burned 
off  in  a  flame.  After  it  has  cooled  the  bacteria  are 
spread  upon  it,  well  diluted  with  water.  After  drying 
thoroughly  in  the  air,  the  stain  is  gradually  poured  on 
and  by  gentle  heating  brought  almost  to  a  boil ;  the  slip 

1  Med.  News,  Sept.  7,  1895. 


104  PATHOGENIC  BACTERIA. 

covered  with  the  hot  stain  is  laid  aside  for  a  minute,  then 
washed  in  water  and  mounted.  In  such  preparations  I 
have  always  been  able  to  see  the  flagella  well,  but  usually 
find  that  while  the  flagella  are  very  distinct,  the  bodies 
of  the  bacteria  are  scarcely  visible. 

Bunge  suggests  a  mordant  consisting  of  a  concentrated 
aqueous  tannin  solution  and  a  i  :  20  solution  of  liq.  ferri 
sesquichloridi  in  water.  The  best  mixture  seems  to  be 
3  parts  of  the  tannin  solution  to  i  part  of  the  diluted 
iron  solution.  To  10  c.cm.  of  this  mixture  i  c.cm.  of  a 
concentrated  aqueous  fuchsin  solution  is  added.  It  is 
not  necessary  to  prepare  this  mordant  fresh  for  each 
staining,  as  it  seems  to  improve  with  age.  The  use  of 
acid  and  alkaline  solutions  added  to  the  mordant  is  dis- 
pensed with. 

The  bacteria  are  carefully  fixed  to  the  glass,  stained 
with  the  mordant  for  five  minutes,  warming  a  little  to- 
ward the  end,  washed,  dried,  and  subsequently  colored 
with  carbol-fuchsin  warmed  a  little. 

Bacteria  can  best  be  measured  by  an  eye-piece  microm- 
eter. As  these  instruments  vary  somewhat  in  con- 
struction, the  unit  of  measurement  for  each  objective 
magnification  or  the  method  of  manipulating  the  adjusta- 
ble instruments  must  be  learned  from  dealers'  catalogues. 

Photographing  bacteria  requires  special  apparatus  and 
methods,  which  are  fully  described  in  text-books  upon 
the  subject. 


CHAPTER  V. 
STERILIZATION  AND  DISINFECTION. 

BEFORE  considering  the  cultivation  of  bacteria  and 
the  preparation  of  media  for  that  purpose  it  is  necessary 
to  discuss  methods  of  destroying  bacteria  whose  acci- 
dental presence  might  ruin  our  experiments. 

The  dust  of  the  atmosphere,  as  has  already  been  shown, 
is  almost  constant  in  its  micro-organismal  contamination, 
so  that  the  spores  of  moulds  and  bacilli,  together  with 
yeasts  and  micrococci,  constantly  settle  from  it  upon  our 
glassware,  enter  our  pots,  kettles,  funnels,  etc.,  and  would 
ruin  every  culture-medium  with  which  we  operate  did 
we  not  take  measures  for  their  destruction. 

Micro-organisms  may  be  killed  by  heat  or  by  the  action 
of  chemicals,  the  processes  being  generically  termed 
sterilization.  The  term  sterilization  is  usually  employed 
to  denote  the  destruction  of  bacteria  by  heat,  in  contra- 
distinction to  disinfection,  which  usually  means  the 
destruction  of  the  bacteria  by  the  use  of  chemical 
agents.  A  chemical  agent  causing  the  death  of  bacteria 
is  called  a  germicide.  An  object  which  is  entirely  free 
from  bacteria  and  their  spores  is  described  as  sterile. 
Certain  substances  whose  action  is  detrimental  to  the 
vitality  of  bacteria  and  prevents  their  growth  amid  other- 
wise suitable  surroundings  are  termed  antiseptics. 

The  study  of  sterilization,  disinfection,  and  antisepsis 
will  naturally  lead  us  through  the  following  subdivisions  : 

I.  The  sterilization  and  protection  of  instruments  and 
glassware  used  in  experimentation. 

II.  The  sterilization  and  protection  of  culture-media. 

III.  The  disinfection  of  the  instruments,  ligatures,  etc. 
and  the  hands  of  the  surgeon,  and  the  use  of  antiseptics. 

IV.  The  disinfection  of  sick-chambers  and  their  con- 
tents, as  well  as  the  dejecta  and  discharges  of  patients 
suffering  from  contagious  and  infectious  diseases. 

105 


106  PA  THOGENIC  BA  CTERIA. 

The  Sterilization  and  Protection  of  Instruments 
and  Glassware  Used  in  Experimentation. — Steriliza- 
tion may  be  accomplished  by  either  moist  or  dry  heat. 
For  the  perfect  sterilization  of  objects  capable  of  with- 
standing it  dry  heat  is  preferable,  because  more  certain 
in  its  action.  If  we  knew  just  what  organisms  we  had 
to  deal  with,  we  might  be  able  in  many  cases  to  save 
time  and  gas,  but  while  some  simple  non-spore-producing 
forms  are  killed  at  a  temperature  of  60°  C.,  others  can 
withstand  boiling  for  an  hour  ;  it  is  therefore  best  to 
employ  a  temperature  high  enough  to  kill  all  with  cer- 
tainty. Platinum  wires  used  for  inoculation  are  held  in 
the  direct  flame  until  they  become  incandescent.  In 
sterilizing  such  wires  attention  must  be  bestowed  upon 
the  glass  handle,  which  should  be  held  in  the  flame  for 
at  least  half  its  length  for  a  few  moments  when  used  for 
the  first  time  each  day.  Carelessness  in  this  respect  may 
cause  the  loss  of  much  time  by  contaminating  cultures. 

Knives,  scissors,  and  forceps  may  be  exposed  for  a  very 
brief  time  to  the  direct  flame,  but  this  affects  the  temper 
of  the  steel  when  continued  too  long.  They  may  also 
be  boiled,  steamed,  or  carbolized. 

All  glassware  is  sterilized  by  exposure  to  a  sufficiently 
hijLjh  temperature,  150°  C.  or  302°  F.,  for  one  hour  in  the 
well-known  hot-air  closet  (Fig.  9).  A  temperature  of 
150°  C.  is  sufficient  to  kill  all  known  bacteria  and  their 
spores  if  continued  for  an  hour. 

Rubber  stoppers,  corks,  wooden  apparatus,  and  other 
objects  which  are  warped,  cracked,  charred,  or  melted 
by  so  high  a  temperature  must  be  sterilized  by  moist 
heat  in  the  steam  apparatus  for  at  least  an  hour  before 
they  can  be  pronounced  sterile. 

It  must  always  be  borne  in  mind  that  after  sterilization 
has  been  accomplished  the  same  sources  of  contamination 
that  originally  existed  are  still  present,  and  begin  to 
operate  as  soon  as  the  objects  are  removed  from  the 
sU-rili/ini;  apparatus. 

To  Schroder   and  Van   Dusch   belong   the   credit   of 


STERILIZATION  AND  DISINFECTION          107 

having  first  shown  that  when  the  mouths  of  flasks  and 
tubes  are  closed  with  plugs  of  sterile  cotton  no  germs 
can  filter  through.  This  observation  has  been  of  ines- 
timable value  to  every  bacteriologist.  Before  sterilizing 


FIG.  9. — Hot-air  sterilizer. 

flasks  and  tubes  we  plug  them  with  ordinary  raw  cotton, 
and  are  sure  that  afterward  their  interiors  will  remain 
free  from  the  access  of  germs  until  opened.  Instruments 
may  be  sterilized  wrapped  in  cotton,  to  be  opened  only 
when  ready  for  use ;  or  instruments  and  rubber  goods 
sterilized  by  steam  can  subsequently  be  wrapped  in 
sterile  cotton  and  kept  for  use.  It  is  of  the  utmost 
importance  to  carefully  protect  every  sterilized  object, 
and  to  allow  as  little  dust  to  collect  upon  it  as  possible, 
in  order  that  the  object  of  the  sterilization  be  not  de- 
feated. As  the  spores  of  moulds  falling  upon  cotton 
sometimes  grow  and  allow  their  mycelia  to  work  their 
way  through  and  drop  into  a  culture-medium,  Roux 


io8 


rATHOGENIC  BACTERIA. 


has  introduced  little  paper  caps  with  which  the  cotton 
stoppers  are  protected  from  the  dust.  These  are  easily 
made  by  curling  a  small  square  of  paper  into  a  "cornu- 
copia, ' '  fastening  by  turning  up  the  edge  or  putting  in  a  pin. 
The  paper  is  placed  over  the  stopper  before  the  sterilization, 
after  which  no  contamination  of  the  cotton  can  occur. 

Sterilization  of  Culture-media. — As  almost  all  of  the 
culture-media  contain  about  80  per  cent,  of  water,  which 
would  be  evaporated  in  the  hot-air  closet,  so  that  the 
material  would  be  destroyed,  hot-air  sterilization  is  not 
appropriate  for  them.  Sterilization  by  streaming  steam 
is  the  best  and  surest  method.  The  prepared  media  are 

placed  in  flasks  or  tubes  care- 
fully plugged  with  cotton  and 
previously  sterilized  with  dry 
heat,  and  then  sterilized  in  what 
is  known  as  Koch's  steam  appa- 
ratus (Fig.  10)  or  in  Arnold's 


i  a-— Koch's  steam  sterilizer. 


FIG.  II.— Arnold's  steam  sterili/.er. 


steam  sterilizer  (Fig.  n),  which  is  more  convenient  and 
more  generally  useful. 

The  temperature  of  boiling  water,   100°  C,  does  not 


STERILIZATION  AND  DISINFECTION. 


109 


kill  many  spores,  so  that  the  exposure  of  culture-media 
to  streaming  steam  is  of  little  use  unless  applied  in 
a  systematic  manner — intermittent  sterilization — based 
upon  a  knowledge  of  sporulation. 

In  carrying  out  the  intermittent  sterilization  the  cul- 
ture-medium is  exposed  for  fifteen  minutes  to  the  passage 
of  streaming  steam  in  the  apparatus  or  to  some  tem- 
perature judged  to  be  sufficiently  high,  so  that  the  bac- 
teria contained  in  it  are  killed.  As  the  spores  remain 
uninjured,  the  medium  is  stood  aside  in  a  cool  place  for 
twenty-four  hours,  and  the  spores  allowed  to  develop  into 
perfect  bacteria. 

When  the  twenty-four  hours  have  passed  the  culture- 
medium  is  again  placed  in  the  apparatus  and  exposed  to 


FIG.  12. — Autoclave  for  rapid  sterilization  FIG.  13. — Kny-Sprague  steam  sterilizer, 
by  superheated  steam  under    pressure. 

the  same  temperature,  until   these  newly-developed  bac- 
teria are  also  killed.      Eventually,  the  process  is  repeated 


1 10  PA  THOGENIC  BACTERIA . 

a  third  time,  lest  a  few  spores  remain  alive  and  capatle 
of  spoiling  the  material.  When  properly  sterilized  in 
this  way,  culture-media  will  remain  free  from  contamina- 
tion until  time  and  evaporation  cause  them  to  dry  up. 


FIG.  14.— Pasteur-Chamberland  filter  arranged  to  filter  under  pressure. 

If  hermetically  sealed,  a  sterile  medium  will  keep  indef- 
initely. 

If  it  should  be  necessary  to  sterilize  culture-media  at 
once,  not  waiting  the  three  days  required  by  the  inter- 
mittent method,  it  may  be  done  by  superheated  steam  in 


STERILIZATION  AND  DISINFECTION. 


II 


an  autoclave  (Fig.  12).  Here  under  a  pressure  of  two  or 
three  atmospheres  sufficient  heat  is  generated  to  destroy 
the  spores.  The  objections  to  this  method  are  that  it 
sometimes  turns  the  agar-agar  dark,  and  that  it  is  likely 
to  destroy  the  gelatinizing  power  of  the  gelatin,  which 
after  sterilization  remains  liquid. 

Liquids  may  also  be  sterilized  by  filtration — i.  e.  by 
passing  them  through  unglazed  porcelain  or  some  other 
material  whose  interstices  are  sufficiently  fine  to  resist  the 
passage  of  the  bacteria.  This  method  is  largely  employed 


FIG.  15. — Kitasato's  filter :  a,  por-  FIG.  16. — Reichel's  bacteriologic  filter 

celain  bougie ;  b,  attachment  for  sue-  of  unglazed  porcelain  :  A^    sterile    re- 

tion-pump;    c,   reservoir;    d,    sterile  ceiver;  B  porcelain  filter ;  c,  <i,  attach- 

receiver.  ments  for  pump. 

for  the  sterilization  of  the  unstable  toxins  and  anti- 
toxins, which  are  destroyed  by  heat.  Various  substances 
have  been  used  for  filtration,  as  stone,  sand,  powdered 
glass,  etc.,  but  experimentation  has  shown  porcelain  to 
be  the  only  reliable  filter  against  bacteria.  Even  this 
material,  whose  interstices  are  so  small  as  to  allow  the 
liquid  to  pass  through  with  great  slowness,  is  only  cer- 
tain in  its  action  for  a  time,  for  after  it  has  been  used 
considerably  the  bacteria  seem  able  to  work  their  way 


112 


PATHOGENIC  BACTERIA. 


through.  To  be  certain  of  the  efficacy  of  such  a  filter 
the  fluid  first  passed  through  must  be  tested  by  cultiva- 
tion methods.  The  complicated  Pasteur-Chamberland 
and  the  simple  Kitasato  and  Reichel  filters  are  shown  in 
Figures  14,  15,  and  16. 

After  having  been  used  a  porcelain  filter  must  be  dis- 
infected, scrubbed,  dried  thoroughly,  and  then  heated  in 
a  Bunsen  burner  or  blowpipe  flame  until  all  the  organic 
matter  is  consumed.  In  this  firing  process  the  filter  first 
turns  black  as  the  organic  matter  chars,  then  becomes 
white  as  it  is  consumed.  The  greatest  care  must  be 
exercised  in  cleansing,  and  especially  must  care  be  taken 
that  the  porcelain  is  dry  before  entering  the  fire,  as  it 
will  certainly  crack  if  moist. 

Before  using  a  new  filter  it  should  be  sterilized  by  dry 
heat,  then  connected  with  receivers  and  tubes,  also  care- 
fully sterilized.  It  should  not  be  forgotten  that  the  fil- 
tered material  is  still  a  good  culture-medium  and  must  be 
handled  with  the  greatest  care. 

While  the  filtration  of  water,  peptone  solution,  and 
bouillon  is  comparatively  easy,  gelatin  and  blood-serum 
pass  through  with  great  difficulty,  and  speedily  gum  the 
filter,  so  that  it  is  useless  until  fired. 

A  convenient  apparatus  used  by  the  author  for  the  rapid 
filtration  of  large  quantities  is  shown  in  the  accompany- 
ing illustration  (Fig.  17). 


FIG.  17. — Apparatus  for  the  rapid  filtration  of  toxins,  etc. 

The  Disinfection  of  Instruments,  Ligatures,  Sutures, 
the  Hands,  etc. — There  are  certain  objects  used  by  the 


STERILIZATION  AND  DISINFECTION.         113 

surgeon  which  cannot  well  be  rendered  incandescent, 
exposed  to  dry  heat  at  150°  C.,  steamed,  or  intermittently 
heated  without  injury.  For  these  objects  disinfection 
must  be  practised.  Ever  since  Sir  Joseph  lyister  intro- 
duced antisepsis,  or  disinfection,  into  surgery  there  has 
been  a  struggle  for  the  supremacy  of  this  or  that  highly- 
recommended  germicidal  substance,  with  two  results — 
viz.  that  a  great  number  of  feeble  germicides  have  been 
discovered,  and  that  belief  in  the  efficacy  of  all  germi- 
cides has  been  somewhat  shaken;  hence  the  origin  of  the 
successful  aseptic  surgery  of  the  present  day,  which 
strives  to  prevent  the  entrance  of  germs  into,  rather  than 
their  destruction  after  admission  to,  the  wound. 

For  a  complete  discussion  of  the  subject  of  antiseptics 
in  relation  to  surgery  the  reader  must  be  referred  to  the 
large  text-books  of  surgery,  where  much  space  is  thus 
occupied.  A  short  list  of  useful  germicides  of  which 
the  respective  values  are  given,  and  a  brief  discussion 
of  some  of  the  more  important  measures,  can  alone  find 
space  in  these  pages.  The  antiseptic  value  of  some  of 
the  principal  substances  used  may  be  expressed  as  fol- 
lows, the  figures  indicating  the  strength  of  the  solution 
necessary  to  prevent  the  development  of  bacteria : 

Pyoktanin  (methyl  violet)  .  i  :  2,000,000 — i  :  5000. 

Formalin i  :  25,000 — i  :  5000. 

Bichlorid  of  mercury    .    .    .  i :  14,3°°. 

Hydrogen  peroxid i :  20,000. 

Formalin  .  i :  20,000. 


Nitrate  of  silver 
Creolin  . 


Chromic  acid 

Thymol 

Salicylic  acid 

Potassium  bichromate  . 

Trikresol 

Zinc  chlorid 


Potassium  permanganate 

Nitrate  of  lead 

Izal  .    .    . 


:  12,500. 

:  5000  to  i  :  200  (does  not  kill 

anthrax). 
15000. 
:  1340. 
:  looo. 
1909. 

:  1000 — i  :  500. 
1526. 

:  285  ;  not  prompt  in  action. 
-.277. 
:  200. 


114  PATHOGENIC  BACTERIA. 


Boracic  acid  .  .  . 
Chloral  hydrate  . 
Ferrous  sulphate 
Calcium  chlorid  . 
Creosote  .  .  . 
Carbolic  acid  .  . 
Alcohol  . 


143- 

107. 

90—1  : 200,  Sternberg. 

25- 

20. 

20  : :  i  :  50. 
10. 


Ether.     Pure  ether  will  not  kill  anthrax  spores  immersed 
in  it  for  eight  days. 

The  value  of  antiseptics,  like  that  of  disinfectants,  is 
always  relative,  the  destructive  as  well  as  the  inhibitory 
power  of  the  solution  varying  with  the  micro-organism 
upon  which  it  acts.  The  following  table,  from  Boer, 
will  illustrate  this : 

Methyl  Violet  (Pyoktaniri). 

Restrains.  Kills. 

Anthrax  bacillus i :  70,000  : 5000 

Diphtheria i :  10,000  :  2000 

Glanders i :  2500  : 150 

Typhoid i :  2500  :  150 

Cholera  spirillum i :  30,000  : 1000 

Large  numbers  of  both  strongly  and  feebly  antiseptic 
substances  have  purposely  been  omitted  from  the  above 
lists,  compiled  from  Sternberg  and  Micquel,  as  either  in- 
appropriate for  ordinary  use  or  as  having  been  replaced 
by  better  agents. 

The  newest,  and  one  of  the  best  germicides  for  all  pur- 
poses is  formaldehyde.  Its  use  as  a  vapor  for  the  sterili- 
zation of  infected  rooms  was  first  suggested  by  Trillat  in 
1895,  but  it  did  not  make  much  stir  in  the  medical  world 
until  a  year  or  more  had  passed  and  a  40  per  cent,  solu- 
tion of  the  gas,  under  the  name  of  "  Formalin,"  had 
been  placed  upon  the  market.  The  original  method  con- 
sisted of  the  evolution  of  the  gas  from  methyl  alcohol  by 
volatilizing  it  in  a  steam  apparatus,  and  passing  the  vapor 
over  a  heated  metal  plate.  At  present  the  original  auto- 
clave has  been  replaced  by  the  apparatus  shown  in  Fig. 
19,  in  which  a  solution  of  formochloral  is  volatilized  by 
heating  under  a  pressure  of  three  atmospheres. 


STERILIZA  TION  AND  DISINFECTION. 


The  gas  is  very  penetrating,  easily  diffusing  itself,  and 
is  said  to  have  enormous  bactericidal  powers.     In  experi- 
ments   conducted    by    Prof. 
Robinson,   of  Bowdoin  Col- 
lege, the  gas  penetrated  mat- 
tresses and  killed  cultures  in 
tubes  wrapped  up  in  them. 

There  seems   to   be   little 
doubt  of  the  ability  of  the 


FIG.  1 8.— The  Trillat  autoclave. 


FlG.  19. — Sanitary  formaldehyde  re- 
generator. 


formaldehyde  gas  to  disinfect,  but  there  are  few  apparatus 
upon  the  market  at  present  that  seem  capable  of  discharg- 
ing a  sufficient  volume  of  the  gas  with  sufficient  rapidity 
to  do  the  work.  The  physician,  therefore,  who  desires 
to  disinfect  with  confidence  should  choose  an  apparatus 
that  has  been  shown  by  competent  experiments  to  fill  the 
requirements. 

The  "formalin,"  or  40  per  cent,  solution  of  the  gas, 
when  fresh  and  tightly  corked,  is  fatal  to  most  bacteria  in 
dilutions  of  from  i :  5000  to  i :  25,000.  It  can  be  employed 
with  great  advantage  to  spray  the  walls  and  floors  of 
rooms.  It  cannot  be  employed  upon  the  skin  or  mucous 
membranes,  because  of  its  marked  irritating  effect. 

The  disinfection  of  the  skin,  both  the  hands  of  the 
surgeon  and  the  part  about  to  be  incised,  is  a  matter  of 
importance.  It  is  almost  impossible  to  secure  absolute 
sterility  of  the  hands,  so  deeply  do  the  skin-cocci  pene- 
trate between  the  layers  of  the  scarf-skin.  The  method  at 


1 16  PA  THOGENIC  BA  CTERIA . 

present  generally  employed,  and  recommended  by  Welch 
and  Hunter  Robb,  is  as  follows:  The  nails  must  be 
trimmed  short  and  perfectly  cleansed.  The  hands  are 
washed  thoroughly  for  ten  minutes  in  water  of  as  high  a 
temperature  as  can  comfortably  be  borne,  soap  and  a  brush 
previously  sterilized  being  freely  used,  and  afterward  the 
excess  of  soap  washed  off  in  clean  hot  water.  The  hands 
are  then  immersed  for  from  one  to  two  minutes  in  a 
warm  saturated  solution  of  permanganate  of  potassium, 
then  in  a  warm  saturated  solution  of  oxalic  acid,  until 
complete  decolorization  of  the  permanganate  occurs,  after 
which  they  are  washed  free  from  the  acid  in  clean  warm 
water  or  salt-solution.  Finally,  they  are  soaked  for  two 
minutes  in  a  i  :  500  solution  of  bichlorid  of  mercury, 
after  which  they  are  ready  for  use. 

Lockwood,1  of  St.  Bartholomew's  Hospital,  recommends 
after  the  use  of  the  scissors  and  penknife,  scrubbing  the 
hands  and  arms  for  three  minutes  in  hot  water  and  soap 
to  remove  all  grease  and  dirt.  The  scrubbing  brush 
ought  to  be  steamed  or  boiled  before  use,  and  kept  in 
i  :  1000  biniodid  of  mercury  solution.  When  the  soap- 
suds have  been  thoroughly  washed  away  with  plenty  of 
clean  water,  the  hands  and  arms  are  thoroughly  washed 
and  soaked  for  not  less  than  two  minutes  in  a  solution  of 
biniodid  of  mercury  in  methylated  spirit;  i  part  of  the 
biniodid  in  500  of  the  spirit.  Hands  that  cannot  bear 
i  :  1000  bichlorid  and  5  per  cent,  carbolic  solutions,  bear 
frequent  treatment  with  the  biniodid.  After  the  spirit 
and  biniodid  have  been  used  for  not  less  than  two  min- 
utes, the  solution  is  washed  off  in  i  :  2000  or  i  :  4000 
biniodid  of  mercury  solution. 

Catgut  cannot  be  sterilized  by  boiling  without  deterio- 
ration. The  present  method  of  preparing  it  is  to  dry  it 
in  a  hot-air  chamber  and  then  boil  it  in  cumol,  which  is 
afterward  evaporated  and  the  skeins  preserved  in  sterile 
test-tubes  or  special  receptacles  plugged  with  sterile  cot- 
ton. Cumol  was  first  introduced  for  this  purpose  by 

1  Brit.  Mcd.  Jour.,  July  II,  1896. 


STERILIZATION  AND  DISINFECTION.         117 

Kronig,  as  its  boiling-point  is  i68°-i78°  C.,  and  thus 
sufficiently  high  to  kill  spores.  The  use  of  cumol  for  the 
sterilization  of  catgut  has  been  carefully  investigated  by 
Clarke  and  Miller.1 

Ligatures  of  silk  and  silkworm-gut  are  boiled  in 
water  immediately  before  using,  or  are  steamed  with  the 
dressings,  or  placed  in  test-tubes  plugged  with  cotton  and 
steamed  in  the  steam  sterilizer. 

At  present,  in  most  hospitals,  instruments  are  boiled 
before  using  in  a  1-2  per  cent,  soda  solution.  Plain 
water  has  the  disadvantage  of  rusting  the  instruments, 
and  during  the  operation  they  are  either  kept  in  the  boiled 
water  or  in  carbolic  solution.  Andrews  makes  special 
mention  of  the  fact  that  the  instruments  must  be  com- 
pletely immersed  to  prevent  rusting. 

During  the  operation  the  wound  is  frequently  washed 
with  normal  salt  solution,  applied  by  sterile  marine  or 
gauze  sponges. 

The  water  and  the  salt  solution  used  for  surgical  pur- 
poses are  to  be  sterilized  before  using,  either  by  steaming 
for  a  prolonged  period,  or  by  the  intermittent  method. 
Large  hospitals  are  generally  furnished  with  special  appa- 
ratus for  supplying  sterile  distilled  water  in  large  quantity. 

To  La  Place  belongs  the  credit  of  observing  that  the 
efficacy  of  bichlorid  of  mercury  is  greatly  increased  by 
the  addition  of  a  small  amount  of  acid,  by  which  the 
penetration  is  increased  and  the  formation  of  insoluble 
albuminates  lessened. 

The  knowledge  that  the  action  of  germicides  is  chem- 
ical, and  that  the  destruction  of  the  bacteria  is  due  to  the 
combination  of  the  germicide  with  the  mycoprotein,  is 
apt  to  lessen  our  confidence  in  the  permanence  of  their 
action.  Geppert  has  shown  of  bichlorid  of  mercury  that 
in  the  reaction  between  it  and  anthrax  spores  the  vitality 
of  the  latter  seems  lost,  but  that  the  precipitation  of  the 
bichlorid  from  this  combination  by  the  action  of  ammo- 
nium sulphid  restores  the  vitality  of  the  spore. 

1  Bull,  of  the  Johns  Hopkins  Hospital,  Feb.  and  March,  1896. 


1 18  PA  THOGENIC  BACTERIA . 

Again,  the  fact  that  some  of  the  antiseptics,  as  nitrate 
of  silver  and  bichlorid  of  mercury,  are  at  once  precipi- 
tated by  albumins,  and  thus  lose  their  germicidal  and 
antiseptic  powers,  limits  the  scope  of  their  employment. 
I  think  it  may  be  safely  said  that  carbolic  acid  is  the 
most  reliable  and  most  generally  useful  of  all  the  germi- 
cides and  antiseptics. 

The  Disinfection  of  Sick-chambers,  Dejecta,  etc. — 
What  has  just  been  remarked  concerning  the  unreliability 
of  many  of  the  germicidal  substances  is  eminently  a 
propos  of  the  disinfection  of  dejecta.  It  is  useless  to 
mix  bichlorid  of  mercury  with  typhoid  stools  or  tubercu- 
lar sputum  rich  in  albumin,  and  imagine  these  substances 
rendered  harmless  in  consequence.  It  should  not  be  for- 
gotten that  the  sick  patient  is  less  the  means  of  convey- 
ing the  contagium  than  the  objects  with  which  he  is  in 
contact,  which  can  be  carried  to  other  rooms  or  houses 
during  or  after  the  progress  of  the  disease.  A  careful 
consideration  of  the  condition  of  the  sick-room  will 
lead  us  to  a  clear  understanding  of  its  bacteriological 
condition. 

The  Air  of  the  Sick-room. — It  is  impossible  to  sterilize 
or  disinfect  the  atmosphere  of  a  room  during  its  occu- 
pancy by  the  patient.  The  disinfecting  capacity  of  the 
solutions  given  above  must  make  obvious  the  concentra- 
tion of  their  useful  solutions,  and  show  the  foolishness 
of  placing  beneath  the  bed  or  in  the  corners  of  a  room 
small  receptacles  filled  with  carbolic  acid  or  chlorinated 
lime.  These  can  serve  no  purpose  for  good,  and  may  be 
potent  for  harm  by  obscuring  the  disagreeable  odors 
emanating  from  materials  which  should  be  removed  from 
the  room  by  the  still  more  disagreeable  odors  of  the  dis- 
infectants. The  practice  of  such  a  custom  is  only  com- 
parable to  the  old  faith  in  the  virtue  of  asafetida  tied 
in  a  corner  of  the  handkerchief  as  a  preventive  of  cholera 
and  smallpox. 

During  the  period  of  illness  a  chamber  in  which  the 
patient  is  confined  should  be  freely  ventilated,  so  that  its 


STERILIZATION  AND  DISINFECTION.          119 

atmosphere  is  constantly  changing  and  replacing  the 
closeness  so  universally  an  accompaniment  of  fever  by 
fresh,  pure  air — a  comfort  to  the  patient  and  a  protection 
to  the  doctors  and  nurses. 

After  recovery  or  death  one  should  rely  less  upon  fu- 
migation than  upon  the  disinfection  of  the  walls  and 
floor,  the  similar  disinfection  of  the  wooden  part  of  the 
furniture,  and  the  sterilization  of  all  else.  The  fumes 
of  sulphur  may  do  some  good — when  combined  with 
steam,  much  good — but  are  greatly  overestimated,  and 
the  sulphurous  vapors  are  rapidly  giving  way  to  the  more 
penetrating  and  germicidal  formaldehyde  vapor.  To 
apply  this,  the  room  to  be  sterilized  is  carefully  closed, 
the  carefully  selected  apparatus  set  in  action,  and  the 
discharged  vapor  allowed  to  act  undisturbed  for  some 
hours,  after  which  the  windows  and  doors  are  all  thrown 
open  to  fresh  air  and  sunlight. 

Instead  of  the  gas,  a  40  per  cent,  solution,  which  can 
be  sprayed  upon  the  ceiling,  walls,  floor,  and  contents  of 
the  room  from  a  large  atomizer,  is  sometimes  used.  Ex- 
perience has  not  shown,  however,  that  this  possesses  any 
distinct  advantages. 

So  far  as  is  at  present  known,  the  disinfection  by  form- 
aldehyde is  complete  and  leaves  nothing  to  be  desired. 
Only  one  point  is  to  be  considered,  already  often  men- 
tioned— that  is,  the  apparatus.  Of  those  experimented 
with  by  the  author,  few  have  given  satisfaction. 

The  Dejecta. — A  little  thought  will  direct  attention  to 
those  of  the  dejections  which  are  dangerous  to  the  com- 
munity and  promote  efforts  for  their  complete  steriliza- 
tion. In  cases  of  diphtheria  the  vomit,  expectorations, 
and  nasal  discharges  are  most  important.  They  should 
be  received  in  old  rags  or  in  Japanese  paper  napkins — 
not  handkerchiefs  or  towels — and  should  be  burned.  The 
sputum  of  tuberculous  patients  should  either  be  collected 
in  a  glazed  earthen  vessel  which  can  be  subjected  to  boil- 
ing and  disinfection,  or,  as  is  an  excellent  plan,  should  be 
received  in  Japanese  rice-paper  napkins,  which  can  at 


120  PATHOGENIC  BACTERIA. 

once  be  burned.     These  napkins  are  not  quite  as  good 
as  the  small  pasteboard  boxes  (Fig.  20)  recommended  by 


FIG.  20.— Pasteboard  cup  for  receiving  infectious   sputum.     When  used  the 
pasteboard  can  be  removed  from  the  iron  frame  and  burned. 

some  city  boards  of  health,  because,  being  highly  absorb- 
ent, the  sputum  is  apt  to  soak  through  and  soil  the  fin- 
gers, etc.  Tuberculous  patients  should  be  provided  with 
rice-paper  instead  of  handkerchiefs,  and  should  have  their 
towels,  knives,  forks,  spoons,  plates,  etc.  kept  strictly 
apart  from  the  others  of  the  household  (though  the  pa- 
tients, whose  mental  acuity  makes  their  sensibilities  very 
pronounced,  need  never  be  told  of  their  isolation),  and 
frequently  boiled  for  considerable  lengths  of  time. 

The  excreta  from  typhoid-fever  and  cholera  cases  re- 
quire particular  attention.  These,  and  indeed  all  alvine 
matter  possibly  the  source  of  infection  or  contagion, 
should  be  received  in  glazed  earthen  vessels  and  imme- 
diately intimately  mixed  with  a  5  per  cent,  solution 
of  chlorinated  lime  (containing  25  per  cent,  of  chlorin) 
if  semi-solid,  or  with  the  powder  if  liquid,  and  allowed 
to  stand  for  an  hour  before  being  thrown  into  the 
drain. 

Tin-  Clothing^  etc. — All  bed-clothing  which  has  been 
used  in  the  sick-room,  all  towels,  napkins,  handkerchiefs, 
nijjht-robes,  underclothes,  etc.  which  have  been  used  by 
the  sick,  and  all  towels,  napkins,  handkerchiefs,  caps, 
aprons,  and  outside  dresses  worn  by  the  nurse,  should  be 
regarded  as  infected  and  subjected  to  sterilization.  The 
only  satisfactory  method  of  doing  this  is  by  prolonged 
subjection  to  steam  in  a  special  apparatus;  but,  as  this 


STERILIZATION  AND  DISINFECTION.         12 1 

is  only  possible  in  hospitals,  the  next  best  thing  is  boiling 
for  some  time  in  the  ordinary  wash-boiler.  When  pos- 
sible, the  clothes  should  be  soaked  in  i :  2000  bichlorid 
solution  before  or  after  boiling,  and  in  drying  should 
hang  in  the  sun  and  wind.  Woollen  underwear  can  be 
treated  exactly  as  if  of  cotton.  The  woollen  clothing  of 
the  patient,  if  infected,  requires  special  treatment.  For- 
tunately, the  infection  of  the  outer  woollen  garments  is 
unusual.  The  only  reliable  method  for  their  purification 
is  prolonged  exposure  to  hot  air  at  110°  C.  In  private 
practice  it  becomes  a  grave  question  what  shall  be  done 
with  these  articles.  Prolonged  exposure  to  fresh  air  and 
sunlight  will  aid  in  rendering  them  harmless  ;  when  it 
is  certain  that  articles  of  wool  are  infected,  they  may  be 
sent  to  the  city  hospital  or  to  certain  of  the  moth-destroy- 
ing and  fumigating  establishments  which  can  be  found 
in  all  large  cities,  and  be  baked. 

The  Furniture,  etc. — The  wholesale  destruction  of  fur- 
niture practised  in  earlier  times  has  at  present  become 
unnecessary.  The  doctor,  if  he  properly  performs  his 
functions,  will  save  much  trouble  and  money  for  his 
patient  by  ordering  the  immediate  isolation  of  his  charge 
in  an  uncarpeted,  scantily-  and  cheaply-furnished  room 
the  moment  an  infectious  disease  is  sitspected,  before 
much  infection  can  have  occurred.  However,  if  before 
his  removal  the  patient  has  occupied  another  bed,  its 
clothing  should  be  promptly  handled  in  the  above- 
described  manner. 

After  the  illness  the  walls  of  the  rooms,  including  the 
ceiling  should  be  sprayed  with  formalin,  or,  where  it  can- 
not be  obtained,  may  be  rubbed  with  fresh  bread,  which 
Loffler  has  shown  to  be  efficacious,  though  scarcely  prac- 
ticable, in  collecting  the  bacteria,  or,  if  possible,  should 
be  whitewashed.  If  the  walls  are  hung  with  paper,  they 
may  be  dampened  with  i  :  1000  bichlorid-of-mercury  so- 
lution before  new  paper  is  hung. 

Aronson1  says:  "For  the  disinfection  of  living-rooms 

lVereinfur  O/fentliche  Gesundheitspflege,  Berlin,  April  26,  1897. 


122  PATHOGENIC  BACTERIA. 

there  is  no  method  that  can  compare  in  the  remotest 
degree,  as  regards  certainty  and  simplicity,  with  that  by 
means  of  formaldehyde  gas.  For  example,  any  one  who 
has  seen  the  process  of  cleansing  walls  by  rubbing  them 
down  with  bread,  as  carried  out  by  the  disinfecting  corps, 
will  agree  with  me  that,  however  effective  it  may  be 
from  a  theoretical  point  of  view,  it  is  absolutely  inefficient 
in  practice.  The  possibility  of  disinfecting  rooms  and  all 
their  contents  with  certainty,  by  means  of  a  simple, 
cheap,  harmless,  and  easily  managed  method  must  be 
hailed  as  a  great  advance." 

The  floor  should  be  scoured  with  5  per  cent,  carbolic- 
acid  solution  or  i  :  1000  bichlorid  of  mercury,  and  all  the 
wooden  articles  wiped  off  two  or  three  times  with  the 
same  solution  employed  for  the  floor.  In  this  scouring 
no  soap  can  be  used,  as  it  destroys  the  virtue  of  the 
germicide.  If  a  straw  mattress  was  used,  it  should  be 
burned  and  the  cover  boiled.  If  a  hair  mattress  was 
used,  it  can  be  steamed  or  baked  by  the  manufacturers, 
who  generally  have  ovens  for  the  purpose.  Curtains, 
shades,  etc.,  should  receive  proper  attention;  but,  of  course, 
the  greater  the  precautions  exercised  in  the  beginning, 
the  fewer  the  articles  which  will  need  attention  in  the 
t-iid.  They  should  be  "removed  before  the  case  has 
developed. 

Strehl  has  succeeded  in  demonstrating  that  when  10  per 
cent,  formalin  solution  is  sponged  upon  artificially  infected 
curtains,  etc.,  the  bacteria  are  killed  by  the  action  of  the 
disinfectant.  This  knowledge  will  be  an  important  ad- 
junct to  our  means  for  disinfecting  the  furniture  of  the 
sick-chamber. 

T/ir  patient,  whether  he  lives  or  dies,  may  also  be 
a  means  of  spreading  the  disease  unless  specially  cared 
for.  After  convalescence  the  body  should  be  bathed  with 
a  weak  bichlorid-of-mercury  solution  or  with  a  2  per 
cent,  carbolic-acid  solution,  or  with  25-50  per  cent,  alco- 
hol, before  the  patient  is  allowed  to  mingle  with  society, 
and  the  hair  should  either  be  cut  off  or  carefully  washed 


STERILIZATION  AND  DISINFECTION          123 

with  the  above  solution.  In  desquamative  diseases  it 
seems  best  to  have  the  entire  body  anointed  with  cos- 
molin  once  daily,  the  unguent  being  well  rubbed  in,  in 
order  to  prevent  the  particles  of  epidermis  being  distrib- 
uted through  the  atmosphere.  Carbolated  cosmolin  may 
be  better  than  the  plain,  not  because  of  the  very  slight 
antiseptic  value  it  possesses,  but  because  it  helps  to  allay 
the  itching  which  may  be  part  of  the  desquamative 
process. 

After  the  patient  is  about  the  room  again,  common 
sense  will  prevent  the  admission  of  strangers  until  all 
the  disinfective  measures  have  been  adopted,  and  after 
this,  touching,  and  especially  kissing  him,  should  be 
omitted  for  some  time. 

The  dead  who  die  of  infectious  diseases  should  be 
washed  in  a  strong  disinfectant  solution,  and  should  be 
given  a  private  funeral  in  which  the  body,  if  exposed, 
should  not  be  touched.  In  my  judgment,  the  body 
is  best  disposed  of  by  cremation. 

It  seems,  however,  to  be  an  error  to  suppose  that  a 
dead  body  can  remain  for  an  indefinite  period  a  source  of 
infection.  Bsmarch l  has  made  a  series  of  laboratory  ex- 
periments to  determine  what  the  fate  of  pathogenic  bac- 
teria in  the  dead  body  really  is.  From  his  results  it  seems 
clear  that  in  septicemia,  cholera,  anthrax,  malignant 
edema,  tuberculosis,  tetanus,  and  typhoid  the  pathogenic 
bacteria  all  die  sooner  or  later,  generally  more  rapidly  in 
conditions  of  decomposition  than  in  good  preservation  of 
the  tissues.  Lack  of  oxygen  may  be  a  cause  of  their 
disappearance. 

1  Zeitschrift  fur  Hygiene,  1893. 


CHAPTER    VI. 
CULTIVATION  OF  BACTERIA;   CULTURE-MEDIA. 

ACCURACY  of  observation  requires  that  the  bacteria  be 
separated  from  their  natural  surroundings  and  artificially 
grown  upon  certain  prepared  media  of  standard  compo- 
sition, in  such  a  manner  that  only  organisms  of  the  same 
kind  are  together. 

One  after  another  various  organic  and  inorganic  mix- 
tures have  been  suggested,  but,  although  almost  any 
compound  containing  organic  matter,  even  in  small 
amounts,  will  suffice  for  the  nourishment  of  bacteria, 
a  certain  few  have  met  with  particular  favor  as  being 
most  valuable. 

Rather  than  give  a  complete  review  of  the  work  which 
has  already  been  done,  in  the  following  pages  the  most 
useful  preparations  only  will  be  considered. 

Our  knowledge  of  the  biology  of  the  bacteria  has 
shown  that  they  grow  best  in  a  mixture  containing  at 
least  80  per  cent,  of  water,  of  a  neutral  or  feebly  alka- 
line reaction,  and  of  a  composition  which,  for  the  patho- 
genic forms  at  least,  should  approximate  the  juices  of 
the  animal  body.  It  might  be  added  that  transparency 
is  a  very  desirable  quality,  and  that  the  most  gener- 
ally useful  culture-media  are  those  that  can  be  readily 
liquefied  and  solidified. 

Bouillon  is  one  of  the  irost  useful  and  most  simple  of 
the  media.  Its  preparation  is  as  follows  :  To  500  grams 
of  finely-chopped  lean,  boneless  beef,  1000  c.cm.  of  clean 
water  are  added  and  allowed  to  stand  for  about  twelve 
hours  on  ice.  At  the  end  of  this  time  the  liquor  is  de- 
canted, that  remaining  on  the  meat  expressed  through  a 
cloth,  and  then,  as  the  entire  quantity  is  seldom  regained, 
HI 


CUL  77  VA  TION  OF  BA  CTERIA .  125 

enough  water  added  to  bring  the  total  amount  up  to  1000 
c.cm.  This  liquid  is  called  the  meat-infusion.  To  it  10 
grams  of  Witte's  or  Fairchild's  dried  beef-peptone  and  5 
grams  of  sodium  chlorid  are  added,  and  the  whole  boiled 
until  the  albumins  coagulate.  The  reaction  is  then  care- 
fully tested,  in  order  that  whatever  sarcolactic  acid  may 
have  been  present  in  the  meat  may  be  neutralized  by  the 
addition  of  a  few  drops  of  a  saturated  aqueous  solution  of 
sodium  carbonate.  The  solution  is  added  drop  by  drop, 
and  the  reaction  frequently  tested  with  litmus-paper. 
When  a  neutral  reaction,  or,  better,  a  faint  alkaline  re- 
action, is  attained,  the  mixture  is  well  stirred,  boiled 
again  for  about  half  an  hour  to  precipitate  the  alkaline 
albumins  formed,  and  filtered.  The  use  of  phenolphtha- 
lein  to  determine  the  reaction  of  the  culture-media  is  much 
more  reliable  than  litmus,  and  in  many  laboratories  has 
replaced  it.  The  method  of  using  it  suggested  by  Timpe 
is  to  continue  the  addition  of  the  carbonate  of  sodium 
solution  until  a  drop  of  it  produces  a  red  spot  upon  phe- 
nolphthalein-paper.  Such  a  paper  can  easily  be  made  by 
using  a  solution  of  5  grams  of  phenolphthalein  to  i  liter 
of  50  per  cent,  alcohol.  The  bibulous  paper  is  cut  into 
strips,  moistened  with  the  solution,  and  then  hung  up  to 
dry.  It  keeps  quite  well.  Acids  do  not  change  the 
appearance  of  the  paper,  but  small  traces  of  alkali  turn 
it  red. 

If  it  is  necessary  to  be  extremely  accurate  concerning 
the  acidity  or  alkalinity  of  the  culture-medium,  the 
method  of  titration  with  phenolphthalein  can  be  em- 
ployed. For  this  purpose  a  small  quantity  of  the  culture- 
fluid — say  10  c.cm. — receives  an  addition  of  a  drop  or 
two  of  a  weak  alcoholic  solution  of  phenolphthalein 
(i  :  300),  and  then  drop  by  drop  from  a  burette  a  dilute 
soda  solution  is  added  until  a  faint  rose  color  occurs,  when 
a  simple  calculation  will  show  that  if  so  much  is  required 
to  bring  about  the  required  color  in  the  10  c.cm.,  so  much 
more  will  be  required  for  the  total  amount.  The  occur- 
rence of  the  rose  color  marks  the  change  from  a  neutral 


126 


•     PATHOGENIC  BACTERIA. 


to  a  faintly  alkaline  reaction.  Definite  varying  degrees 
of  alkalinity  can  be  secured  by  adding  measured  quan- 
tities of  the  soda  solution  beyond  that  necessary  to  bring 
about  the  beginning  alkalinity.  In  using  phenolphtha- 
lein  one  should  remember  that  the  first  sign  of  rose  color 
marks  a  change  to  alkalinity,  and  that  this  is  a  higher 
degree  of  alkalinity  than  that  required  to  turn  red  litmus 
blue. 

The  bouillon  thus  prepared  is  a  clear  fluid  of  a  straw 
color,  much  resembling  normal  urine  in  appearance.     It 


21.— Funnel  for  filling  tubes  with  culture-media  (Warren). 

is  dispensed  in  previously  sterilized  tubes  with  cotton 
plugs—about  10  c.cm.  to  each— and  is  then  sterilized  by 
steam  three  successive  days  for  fifteen  to  twenty  minutes 
each,  according  to  the  directions  already  given  for  frac- 
tional sterilization.  (Seep.  109.) 


CUL  77  VA  TION  OF  BA  CTERIA .  127 

For  the  preparation  of  bouillon,  as  well  as  gelatin, 
agar-agar,  and  glycerin  agar  still  to  be  described,  beef- 
extract  (Liebig's)  may  be  employed,  but  for  the  most 
delicate  work  this  is  rather  undesirable,  because  of  its 
unstable  composition  and  because  of  the  precipitation  of 
meat-salts,  which  can  scarcely  be  filtered  out  of  the  agar- 
agar,  owing  to  the  fact  that  they  only  crystallize  when 
the  solution  cools.  When  it  is  desirable  to  prepare  the 
bouillon  from  beef-extract,  the  method  is  very  simple. 
To  1000  c.cm.  of  clean  water  10  grams  of  Whitte's  dried 
beef-peptone,  5  grams  of  sodium  chlorid,  and  about  2 
grams  of  beef-extract  are  added.  The  solution  is  boiled 
until  the  constituents  are  dissolved,  neutralized,  if  neces- 
sary, and  filtered  when  cold.  If  it  is  filtered  while  hot, 
there  is  always  a  subsequent  precipitation  of  meat-salts, 
which  clouds  it. 

Bouillon  and  other  liquid  culture-media  are  best  dis- 
pensed and  kept  in  small  receptacles — test-tubes  or  flasks 
—in  order  that  a  single  contaminating  organism,  should 
it  enter,  may  not  spoil  the  entire  bulk.  A  very  con- 
venient simple  apparatus  used  by  bacteriologists  for  fill- 
ing tubes  with  liquid  media  is  shown  in  Figure  21.  It 
need  not  be  sterilized  before  using,  as  the  culture-medium 
will  be  sterilized  by  the  intermittent  method  after  the 
tubes  are  filled.  The  test-tubes  and  flasks  into  which 
the  culture-medium  is  filled  must,  however,  be  previously 
sterilized  by  dry  heat.  The  dry-heat  sterilization  is  done, 
of  course,  after  the  cotton  plugs  are  in  place. 

Bouillon  is  the  basis  of  most  of  the  culture-media. 
The  addition  of  10  per  cent,  of  gelatin  makes  it  "gela- 
tin ;"  that  of  i  per  cent,  of  agar-agar  makes  it  "agar- 
agar.  ' '  The  preparation  of  these  media,  however,  varies 
somewhat  from  that  of  the  plain  bouillon. 

Gelatin. — The  culture-medium  known  as  gelatin  has  de- 
cided advantages  over  the  bouillon,  not  only  because  it  is 
an  excellent  food  for  bacteria,  and,  like  the  bouillon,  trans- 
parent, but  because  it  is  also  solid.  Nor  is  this  all  :  it  is 
a  transparent  solid  which  can  be  made  liquid  or  solid  at 


128  PATHOGENIC  BACTERIA. 

will.  It  is  prepared  as  follows  :  To  1000  c.cm.  of  meat- 
infusion  or  to  1000  c.cm.  of  water  containing  2  grains  of 
beef-extract  in  solution,  10  grams  of  peptone,  5  grains  of 
salt,  and  100  grams  of  gelatin  ("Gold  label "  is  the  best 
commercial  article)  are  added,  and  boiled  for  about  an 
hour  over  a  moderately  hot  flame.  Double  boilers  are 
very  slow,  and  if  proper  care  is  exercised  there  is  little 
danger  of  the  gelatin  burning.  It  must  be  stirred  occa- 
sionally, and  the  flame  should  be  so  distributed  by  wire 
gauze  as  not  to  act  upon  a  single  point  of  the  bottom  of 
the  kettle.  At  the  end  of  the  hour  the  albumins  of 
the  meat-infusion  will  be  coagulated  and  the  gelatin 
thoroughly  dissolved.  Giinther  has  shown  that  the 
gelatin  congeals  better  if  allowed  to  dissolve  slowly  in 
warm  water  before  boiling.  The  liquid  is  now  cooled 
to  60°  C.  and  neutralized — i.  e.  alkalinized.  As  the  gela- 
tin is  itself  acid,  a  relatively  larger  amount  of  the  sodium- 
carbonate  solution  will  be  needed  than  was  required  for 
the  bouillon.  When  the  proper  reaction  is  attained,  as 
much  water  as  has  been  lost  by  vaporization  during  the 
process  of  boiling,  intimately  mixed  with  the  white  of 
an  egg,  is  added,  well  stirred  in,  and  the  whole  boiled 
for  half  an  hour,  then  filtered. 

If  the  filter- paper  be  of  good  quality  and  properly 
folded  (pharmaceutical  filter),  and  if  the  gelatin  be  prop- 
erly dissolved,  the  whole  quantity  should  pass  through 
before  cooling  too  much.  Should  only  half  go  through 
before  cooling,  the  remainder  must  be  returned  to  the 
pot,  heated  to  boiling  once  more,  and  then  passed  through 
a  new  filter-paper.  As  a  matter  of  fact,  gelatin  generally 
filters  readily.  A  wise  precaution  is  to  catch  the  first  few 
centimeters  in  a  test-tube  and  boil  them,  so  that  if  a 
cloudiness  shows  the  presence  of  uncoagulated  albumin, 
the  whole  mass  can  be  boiled  again.  The  finished  gel- 
atin is  at  once  distributed  into  sterilized  tubes,  and  then 
sterilized  like  the  bouillon  by  the  fractional  method. 

Of  course,  the  gelatin  or  any  other  culture-medium  can 
be  kept  en  masse  indefinitely,  but  should  a  contaminating 


CUL  77  VA  TJON  OF  BA  CTERIA .  129 

micro-organism  accidentally  enter,  the  whole  quantity 
will  be  spoiled  ;  if,  on  the  other  hand,  it  is  kept  in  tubes, 
several  of  them  may  be  lost  without  much  inconvenience. 
Under  proper  precautions  of  sterilization  and  protection 
it  should  all  keep  well. 

Agar-agar. — Agar-agar  is  the  commercial  name  of  a 
Japanese  sea-weed  which  dissolves  in  boiling  water  with 
resulting  thick  jelly  when  cold.  The  jelly,  which  solidi- 
fies between  30°  and  40°  C.,  cannot  again  be  melted  ex- 
cept by  the  elevation  of  its  temperature  to  the  boiling- 
point,  so  that  this  culture-medium,  which  is  nearly  trans- 
parent, is  almost  as  useful  as  gelatin.  In  addition  to  its 
readiness  to  liquefy  and  solidify,  it  is  sufficiently  firm 
to  allow  of  the  incubation-temperature — i.  e.  37°  C. — at 
which  gelatin  is  always  liquid,  and  no  better  than  bouillon. 

The  preparation  of  this  medium  is  generally  described 
in  the  text-books  as  one  u  requiring  considerable  patience 
and  much  waste  of  filter- paper. n  In  reality,  it  is  not  dif- 
ficult if  a  good  heavy  filter-paper  be  obtained  and  no 
attempt  be  made  to  filter  the  solution  until  the  agar-agar 
is  perfectly  dissolved.  It  is  prepared  as  follows  :  To  1000 
c.cm.  of  bouillon  made  as  described  above,  preferably  of 
meat  instead  of  beef-extract,  10  grams  of  agar-agar  are 
added.  The  mixture  is  boiled  for  an  hour,  or,  if  possible, 
two.  At  the  end  of  the  first  hour  it  is  cooled  to  about 
60°  C.,  and  after  neutralization,  which  may  not  be  neces- 
sary if  the  bouillon  was  neutral,  an  egg  beaten  up  in 
water  is  added,  and  the  liquid  is  boiled  again  until  the 
egg  is  entirely  coagulated.  The  reaction  of  the  agar-agar 
should  be  neutral  rather  than  alkaline,  as,  for  an  un- 
known reason,  alkalinity  seems  to  interfere  slightly  with 
filtration. 

After  the  boiling,  which  should  be  brisk,  has  caused 
the  thorough  solution  of  the  agar-agar,  it  is  filtered,  just 
as  the  gelatin  was,  through  a  carefully-folded  pharmaceu- 
tical filter  wet  with  boiling  water.  It  may  expedite  mat- 
ters to  pour  in  about  one-half  of  the  solution,  keep  the 
remainder  hot,  and  subsequently  add  it  when  necessary. 

9 


130  PATHOGENIC  BACTERIA. 

Experience  shows  that  1000  c.cm.  of  agar-agar  rarely  go 
•through  one  paper,  and  I  always  expect  when  beginning 
the  filtration  to  be  compelled  to  boil  the  material  which 
remains  on  the  paper  again,  and  pour  it  through  a  new 
filter. 

The  formerly  much-employed  hot-water  and  gas-jet 
filters  seem  unnecessary.  If  properly  prepared,  the  whole 
quantity  will  filter  in  from  fifteen  to  thirty  minutes. 

If  made  from  beef-extract,  the  agar-agar  almost  always 
precipitates  a  considerable  amount  of  meat-salts  as  it 
cools.  This  should  be  anticipated,  but,  so  far  as  I  can 
determine,  cannot  always  be  prevented.  The  amount  is 
certainly  lessened  by  making  the  bouillon  first,  filtering 
it  cold,  then  adding  the  agar-agar,  and  dissolving  and 
filtering  it. 

The  difficulty  of  filtering  the  agar-agar  has  led  Fliigge 
and  others  to  adopt  a  method  of  sedimentation.  An  in- 
genious apparatus  for  this  purpose  has  lately  been  devised 
by  Bleisch.  The  methods  can  be  simplified  by  using  a 
small  pharmaceutical  percolator,  the  bottom  of  which  is 
closed  by  a  rubber  cork  containing  a  tube  which  extends 
nearly  to  the  top  of  the  percolator  and  is  attached  to 
a  rubber  tube  with  a  pinchcock  below.  The  melted  agar- 
agar  is  poured  into  this,  and  kept  in  the  steam  apparatus 
until  the  sedimentation  is  sufficient  to  allow  clear  fluid  to 
be  drawn  from  the  top.  As  the  clear  agar-agar  is  drawn 
off  the  tube  is  pulled  down  through  the  rubber  cork,  and 
more  drawn  off  until  only  the  sediment  is  left. 

Agar-agar  is  dispensed  in  tubes  like  the  gelatin  and 
bouillon,  sterilized  by  steam  by  the  intermittent  process, 
and  after  the  last  sterilization,  before  cooling,  each  tube 
is  inclined  against  a  slight  elevation,  so  as  to  offer  an  ex- 
tensive flat  surface  for  the  culture. 

After  the  agar-agar  jelly  solidifies  its  contraction  causes 
some  water  to  collect  at  the  lower  part  of  the  tube.  This 
should  not  be  removed,  as  it  keeps  the  material  moist, 
and  also  because  it  has  a  distinct  influence  upon  the  cha- 
racter of  the  growth  of  the  bacteria. 


CULTIVATION  OF  BACTERIA.  131 

Glycerin  Agar-agar. — For  an  unknown  reason  certain 
of  the  bacteria  which  will  not  grow  upon  the  agar-agar 
as  prepared  above  will  do  so  if  3-7  per  cent,  of  glycerin 
be  added.  Among  these  is  the  tubercle  bacillus,  which, 
not  growing  at  all  upon  plain  agar-agar,  will  grow  well 
when  glycerin  is  added — a  fact  discovered  by  Roux  and 
Nocard.  The  glycerin  may  also  be  added  to  gelatin  or 
any  other  medium. 

Blood  Agar-agar  was  recommended  by  R.  Pfeiffer  for 
the  cultivation  of  the  influenza  bacillus.  It  is  ordinary 
agar-agar  whose  surface  is  coated  with  a  little  blood 
secured  under  antiseptic  precautions  from  the  finger-tip, 
ear-lobule,  etc.,  of  man,  or  the  veins  of  one  of  the  lower 
animals.  Some  bacteriologists  prepare  a  hemoglobin 
agar-agar  by  spreading  a  little  powdered  hemoglobin 
upon  the  surface  of  the  agar-agar.  This  has  the  disad- 
vantage that  powdered  hemoglobin  is  not  sterile,  and  the 
medium  must  be  sterilized  after  its  addition. 

The  blood  agar-agar  should  be  kept  in  the  incubator  a 
day  or  two  before  use  so  as  to  insure  perfect  sterility. 

Blood-serum. — The  great  advantage  possessed  by  this 
medium  is  that  it  is  itself  a  constituent  of  the  body,  and 
hence  offers  opportunities  for  the  development  of  the 
parasitic  forms  of  bacteria  under  the  most  natural  con- 
ditions possible.  It  is  the  most  difficult  of  all  the  media 
to  prepare.  The  blood  must  be  obtained  from  a  slaughter- 
house in  an  appropriate  receptacle,  the  best  things  for  the 
purpose  being  tall  narrow  jars  of  about  i  liter  capacity, 
with  a  tightly-fitting  lid.  The  jars  are  sterilized  by  heat 
or  by  washing  with  alcohol  and  ether,  are  carefully  dried, 
closed,  and  carried  to  the  slaughter-house  where  the  blood 
is  to  be  obtained.  As  the  blood  flows  from  the  severed 
vessels  of  the  animal  the  jars  are  filled  one  by  one.  It 
seems  advisable  to  allow  the  first  blood  to  escape,  as  it  is 
likely  to  become  contaminated  from  the  hair.  By  waiting 
until  a  coagulum  forms  upon  the  hair  the  danger  of  con- 
tamination is  obviated.  The  jars  when  full  are  allowed 
to  stand  undisturbed  until  quite  firm  coagula  form  within 


132  PATHOGENIC  BACTERIA. 

them.  If  these  have  any  tendency  to  cling  to  the  glass, 
each  one  should  be  given  a  few  violent  twists,  so  as  to 
break  away  the  fibrinous  attachments.  After  this  the 
jars  are  carried  to  the  laboratory  and  stood  upon  ice  for 
forty-eight  hours,  by  which  time  the  clots  will  have  re- 
tracted considerably,  and  a  moderate  amount  of  clear 
serum  can  be  removed  by  sterile  pipettes  and  placed  in 


FIG.  22. — Koch's  apparatus  for  coagulating  and  sterilizing  blood-serum. 


sterile  tubes.  If  the  serum  obtained  is  red  and  clouded 
from  the  presence  of  corpuscles,  it  may  be  pipetted  into 
sterile  cylinders  and  allowed  to  sediment  for  twelve  hours, 
then  repipetted  into  tubes.  It  is  evident  that  such  com- 
plicated maneuvring  will  offer  many  possible  chances  of 
infection  ;  hence  the  sterilization  of  the  serum  is  of  the 
greatest  importance. 

If  it  is  desirable  to  use  the  serum  as  a  liquid  medium,  it 
is  exposed  to  a  temperature  of  60°  to  65°  C.  for  one  hour 
upon  each  of  five  consecutive  days.  If  it  is  thought  best 
to  coagulate  the  serum  and  make  a  solid  culture-medium, 
it  may  be  exposed  twice,  for  an  hour  each  time — or  three 
times  if  there  is  distinct  reason  to  think  it  contam- 
inated— to  a  temperature  just  short  of  the  boiling-point. 
During  the  process  of  coagulation  the  tubes  should  be 
inclined,  so  as  to  offer  a  large  surface  for  the  growth  of 


CUL  77  VA  TION  OF  BA  CTERTA .  133 

the  culture.  The  serum  thus  prepared  may  be  white,  or 
have  a  reddish-gray  color  if  many  corpuscles  are  pres- 
ent, and  is  opaque.  It  cannot  be  melted,  but  once  solid 
remains  so. 

Koch  devised  a  very  good  apparatus  (Fig.  22)  for  coag- 
ulating blood-serum.  The  bottom  should  be  covered 
with  cotton,  a  single  layer  of  tubes  placed  upon  it,  and 
the  temperature  elevated  until  coagulation  occurs.  The 
repeated  sterilizations  may  be  conducted  in  this  apparatus, 
or  may  be  done  equally  well  in  the  steam  apparatus,  the 
cover  of  which  is  not  completely  closed,  for  if  the  tem- 
perature of  the  serum  is  raised  too  high  it  is  certain  to 
bubble. 

Loffler's  blood-serum  mixture,  which  seems  rather 
better  for  the  cultivation  of  some  species  than  the  blood- 
serum  itself,  consists  of  i  part  of  a  beef-infusion  bouillon 
containing  i  per  cent,  of  glucose  and  3  parts  of  liquid 
blood-serum.  After  being  well  mixed  this  is  distributed 
in  tubes,  and  sterilized  and  coagulated  like  the  blood- 
serum  itself.  Most  organisms  grow  more  luxuriantly 
upon  it  than  upon  either  plain  blood-serum  or  other 
culture-media.  Its  special  usefulness  is  for  the  Bacillus 
diphtherise,  which  grows  upon  it  with  rapidity  and  with 
quite  a  characteristic  appearance. 

Alkaline  Blood-serum. — According  to  Lorrain  Smith, 
a  very  useful  culture-medium  can  be  prepared  as  follows: 
To  each  100  c.cm.  of  blood-serum  add  1-1.5  c.cm.  of  a  10 
per  cent,  solution  of  sodium  hydrate  and  shake  it  gently. 
Put  sufficient  of  the  mixture  into  each  of  a  series  of  test- 
tubes,  and,  laying  them  upon  their  sides,  sterilize  like 
blood-serum,  taking  care  that  their  contents  are  not 
heated  too  quickly,  as  then  bubbles  are  apt  to  form. 
The  result  should  be  a  clear,  solid  medium  consisting 
chiefly  of  alkali-albumins.  It  is  especially  useful  for 
the  bacillus  diphtheriae. 

Deycke's  Alkali-albuminate. — 1000  grams  of  meat  are 
macerated  twenty-four  hours  with  1200  c.cm.  of  a  3  per 
cent,  solution  of  potassium  hydrate.  The  clear  brown  fluid 


134  PATHOGENIC  BACTERIA. 

is  filtered  off  and  pure  hydrochloric  acid  carefully  added 
while  a  precipitate  forms.  The  precipitated  albuminate 
is  collected  upon  a  cloth  filter,  mixed  with  a  small  quan- 
tity of  liquid,  and  made  distinctly  alkaline.  To  make 
solutions  of  it  of  definite  strength  it  can  be  dried,  pul- 
verized, and  redissolved. 

The  most  useful  formula  used  by  Deycke  was  a  2^  per 
cent,  solution  of  the  alkali-albuminate  with  i  per  cent,  of 
peptone,  i  per  cent,  of  NaCl,  and  gelatin  or  agar-agar 
enough  to  make  it  solid. 

Potatoes. — Without  taking  time  to  review  the  old 
method  of  boiling  potatoes,  opening  them  with  sterile 
knives,  and  protecting  them  in  the  moist  chamber,  or 
the  much  more  easily  conducted  method  of  Esmarch  in 
which  the  slices  of  potato  are  sterilized  in  the  small 
dishes  in  which  they  are  afterward  kept  and  used,  we 
will  at  once  pass  to  what  seems  the  most  simple  and 
satisfactory  method  of  using  this  valuable  medium — that 
of  Bolton  and  Globig:1 

With  the  aid  of  a  cork-borer  a  little  smaller  in  diam- 
eter than  the  test-tube  ordinarily  used  a  number  of  cyl- 
inders are  cut  from  potatoes.  Rather  large  potatoes 
should  be  used,  the  cylinders  being  cut  transversely,  so 
that  a  number,  each  about  an  inch  and  a  half  in  length, 
can  be  cut  from  one  potato.  The  skin  is  removed  from 
the  cylinders  by  cutting  off  the  ends,  after  which  each 
cylinder  is  cut  in  two  by  an  oblique  incision,  so  as  to 
leave  a  broad,  flat  surface.  The  half-cylinders  are  placed 
each  in  a  test-tube  previously  sterilized,  and  then  are 
exposed  three  times,  for  half  an  hour  each,  to  the  pass- 
ing steam  of  the  sterilizer.  This  steaming  cooks  the 
potato  and  also  sterilizes  it.  Such  cultures  are  apt  to 
deteriorate  rapidly,  first  by  turning  very  dark ;  second, 
by  drying  so  as  to  be  useless.  Abbott  has  shown  that 
if  the  cut  cylinders  be  allowed  to  stand  for  twelve  hours 
in  running  water  before  being  dispensed  in  the  tubes, 
tlu\  do  not  turn  dark.  Drying  may  be  prevented  by 

1  The  Medical  News,  vol.  1.,  1887,  p.  138. 


CULTIVATION  OF  BACTERIA.  135 

adding  a  few  drops  of  clean  water  to  each  tube  before 
sterilizing.  It  is  not  necessary  to  have  a  special  small 
chamber  blown  in  the  tube  to  contain  this  water ;  only 
a  small  quantity  need  be  added,  and  this  will  not  touch 
the  potato,  which  does  not  reach  the  bottom  of  the 
rounded  tube. 

A  potato-juice  has  also  been  suggested,  and  is  of  some 
value.  It  is  made  thus  :  To  300  c.cm.  of  water  100  grams 
of  grated  potato  are  added,  and  allowed  to  stand  on  ice 
over  night.  Of  the  pulp  300  c.  cm.  are  expressed  through 
a  cloth  and  cooked  for  an  hour  on  a  water-bath.  After 
cooking,  the  liquid  is  filtered  and  receives  4  per  cent,  of 
glycerin.  It  may  or  may  not  need  neutralization.  Upon 
this  medium  the  tubercle  bacillus  grows  well,  especially 
when  the  reaction  of  the  medium  is  acid,  but  loses  its 
virulence. 

Milk. — Milk  is  useful  as  a  culture-medium.  As  when 
the  milk  stands  the  cream  which  rises  to  the  top  is  a 
source  of  inconvenience,  it  is  best  to  secure  from  a  dairy 
fresh  milk  from  which  the  cream  has  been  removed  by 
a  centrifugal  machine.  It  is  placed  in  sterile  tubes  and 
sterilized  by  steam  by  the  intermittent  method.  The 
opaque  nature  of  this  culture-medium  often  permits  the 
undetected  development  of  contaminating  organisms. 
A  careful  watch  should  therefore  be  kept  upon  it  lest  it 
spoil. 

Litmus  Milk. — This  is  milk  to  which  just  enough  of 
a  saturated  watery  solution  of  pulverized  litmus  is  added 
to  give  a  distinct  blue  color.  Cow's  milk  is  inclined  to 
be  acid  in  reaction,  and  a  small  amount  of  sodium  car- 
bonate may  be  necessary  to  give  it  a  distinct  blue.  The 
use  of  litmus  is  probably  the  best  method  of  determining 
whether  bacteria  by  their  growth  produce  acids  or  alka- 
lies. 

The  watery  solution  of  litmus,  being  a  vegetable  in- 
fusion, is  likely  to  spoil;  hence  it  should  always  be  treated 
like  the  culture-media  and  sterilized  by  steam  every  time 
the  receptacle  in  which  it  is  kept  is  opened. 


136  PATHOGENIC  BACTERIA. 

Petruschky's  Whey. — In  order  to  differentiate  be- 
tween acid  and  alkaline  producers  among  the  bacteria, 
Petruschky  has  recommended  a  neutral  whey  colored 
with  litmus.  It  is  made  as  follows: 

To  a  liter  of  fresh  skimmed  milk  i  liter  of  water  is 
added.  The  mixture  is  violently  shaken.  About  10  c.cm. 
are  now  taken  out  as  a  sample  to  determine  how  much 
hydrochloric  acid  must  be  added  to  produce  coagulation 
of  the  milk,  and,  having  determined  the  least  quantity 
required  for  the  whole  bulk,  it  is  added.  After  coagulation 
the  whey  is  filtered  off,  exactly  neutralized  and  boiled. 
After  boiling  it  is  generally  found  clouded  and  acid  in 
reaction.  It  is  therefore  filtered  again,  and  again  neu- 
tralized. Litmus  is  finally  added  to  the  neutral  liquid,  so 
that  it  has  a  violet  color,  which  can  readily  be  changed  to 
blue  or  red  by  alkalies  or  acids. 

The  medium  is  a  very  useful  aid  in  differentiating 
the  typhoid  and  colon  bacilli,  showing  well  the  alkaline 
formation  of  the  typhoid  bacillus. 

Peptone  Solution,  or  Dunham's  solution,  is  very  use- 
ful for  the  detection  of  certain  faint  colors.  It  is  a  per- 
fectly clear,  colorless  solution,  made  as  follows: 

Sodium  chlorid,  0.5^  Boil  until  the  ingredients 

Witte's  dried  peptone,    i.  dissolve;  then  filter,  fill 

Water,  100.    J      into  tubes,  and  sterilize. 

It  is  one  of  the  best  media  for  the  detection  of  indol. 
In  it  the  bacillus  pyocyaneus  produces  its  blue  color.  A 
very  important  fact  in  regard  to  peptone  has  been  pointed 
out  by  Garini,1  who  found  that  many  of  the  peptones 
upon  the  market  were  impure,  and  on  this  account  failed 
t<>  show  the  indol  reaction  for  bacteria  known  to  produce 
indol.  He  recommends  the  use  of  the  biuret  reaction 
for  testing  the  peptone  to  be  employed.  The  reagent 
used  is  Fehling's  copper  solution,  with  which  pure  pep- 
tone strikes  a  violet  color  not  destroyed  upon  boiling, 

1  Ctntralbl.f.  Bakt.  u.  Parasitcnk.,  1893,  xiii.,  p.  790. 


CULTIVATION  OF  BACTERIA.  137 

while  impure  peptone  gives  a  red  or  reddish-yellow  pre- 
cipitate. Both  the  peptone  and  copper  solution  should 
be  in  a  dilute  form  to  make  successful  tests.  The 
addition  of  4  c.cm.  of  the  following  solution — 

Rosalie  acid,  0.5, 

80  per  cent,  alcohol,  100. 

makes  it  become  an  excellent  reagent  for  the  detection 
of  acids  and  alkalies.  The  solution  is  pale  rose  in  color. 
If  the  bacterium  produces  acids,  the  color  fades;  if  alka- 
lies, it  intensifies.  As  the  color  of  rosalic  acid  is  destroyed 
by  glucose,  it  cannot  be  used  in  culture-media  contain- 
ing it. 

Theobald  Smith  calls  attention  to  the  fact  that  Dun- 
ham's solution  is  unsuited  to  the  growth  of  many  bac- 
teria, some  failing  altogether  to  grow  in  it,  and  recom- 
mends that,  instead,  bouillon  free  of  dextrose  shall  be  used. 
All  bacteria  grow  well  in  it,  and  the  indol-reaction  is 
pronounced  in  sixteen-hour-old  cultures.  His  method  of 
preparation1  is  as  follows:  beef-infusion,  prepared  either 
by  extracting  in  the  cold  or  at  60°  C.,  is  inoculated  in 
the  evening  with  a  rich  fluid  culture  of  some  acid-pro- 
ducing bacterium  (Bacillus  coli),  and  placed  in  the  ther- 
mostat. Early  next  morning  the  infusion,  covered  with 
a  thin  layer  of  froth,  is  boiled,  filtered,  peptone  and  salt 
added  and  the  neutralization  and  sterilization  carried  on 
as  usual, 

To  test  for  the  presence  of  indol,  the  bacterium  is 
planted  in  the  culture-medium,  allowed  to  grow  for 
upward  of  twelve  hours,  and  then  subjected  to  the  com- 
bined action  of  a  nitrite  and  chemically  pure  sulphuric 
acid.  In  making  the  test,  Smith  adds  to  each  tube  i  c.cm. 
of  a  o.oi  per  cent,  solution  of  KNO2,  freshly  prepared, 
and  10  drops  of  chemically  pure  H2SO4.  The  presence 
of  indol  is  characterized  by  the  production  of  a  red 
color. 

1  Journal  of  Exp.  Medicine,  vi.}  Sept.  5,  1897,  p.  546. 


138  PATHOGENIC  BACTERIA. 

It  is  not  intended  that  the  student  shall  infer  that 
there  are  no  culture-media  other  than  these,  which  have 
been  selected  because  of  their  usefulness  and  popularity. 
Many  other  compounds  and  as  many  simple  substances 
are  employed ;  for  example,  eggs,  white  of  egg,  urine, 
bread,  sputum,  sugar  solutions,  hydrocele  fluid,  and 
aqueous  humor. 


CHAPTER   VII. 
CULTURES,  AND  THEIR  STUDY. 

THE  objects  which  we  have  had  before  us  in  the  prep- 
aration of  the  culture-media  were  numerous.  We  have 
prepared  them  so  as  to  allow  us  to  separate — or,  rather, 
to  isolate — bacteria,  to  keep  them  in  healthy  growth  for 
considerable  lengths  of  time,  to  enable  us  to  observe  their 
biologic  peculiarities,  and  to  introduce  them  without  dif- 
ficulty into  the  bodies  of  animals. 

The  isolation  of  bacteria  was  impossible  until  the  fluid 
culture-media  of  the  early  observers  were  replaced  by  the 
solid  media,  and  was  exceedingly  crude  until  Koch  gave 
us  the  solid,  transparent  media  and  the  well-known 
"  plate-cultures." 

A  growth  of  artificially-planted  micro-organisms  in 
which  an  immense  number  are  massed  together  is  called 
a  culture.  If  such  a  growth  contains  but  one  kind  of 
organism,  it  is  known  as  a  pure  culture. 

It  has  become  the  habit  at  present  to  use  the  term  "cul- 
ture" rather  loosely,  so  that  it  does  not  always  signify  a 
growth  of  micro-organisms  artificially  planted,  but  may 
signify  a  growth  taking  place  under  natural  conditions ; 
thus,  typhoid  bacilli  are  said  to  exist  in  the  spleens  of 
patients  dead  of  that  disease  "in  pure  culture,"  because 
no  other  bacteria  are  there  ;  and  sometimes,  when  in  ex- 
pectorated fragments  of  cheesy  matter  from  tuberculosis 
pulmonalis  the  tubercle  bacilli  are  very  numerous  and 
unmixed  with  other  bacteria,  the  term  "pure  culture" 
is  again  used  to  describe  the  condition. 

Three  principal  methods  are  at  present  employed  to 
enable  us  to  secure  pure  cultures  of  bacteria,  but  before 
beginning  a  description  of  them  it  is  well  to  observe  that 


139 


140 


PATHOGENIC  BACTERIA. 


the  peculiarities  of  certain  pathogenic  forms  enable  us 
to  use  special  means,  taking  advantage  of  their  eccentrici- 
ties, for  their  isolation,  and  that  the  general  methods  are 
in  reality  more  useful  for  the  non-pathogenic  than  for  the 
pathogenic  forms. 

All  three  methods  depend  upon  the  observation  of 
Koch,  that  when  germs  are  equally  distributed  through- 
out some  liquefied  nutrient  medium  which  can  be  solidi- 
fied in  a  thin  layer,  the  growth  of  the  germs  takes  place 
in  little  scattered  groups  or  families,  called  colonies,  dis- 
tinctly separated  from  each  other  and  capable  of  trans- 
plantation to  tubes  of  culture-media. 

Plate-cultures.— The  plate-cultures,  originally  made 
by  Koch,  require  considerable  apparatus,  and  of  late  years 
have  given  place  to  the  more  ready  methods  of  Petri  and 
Von  Esmarch.  So  great,  however,  is  the  historic  interest 
attached  to  the  plates  that  it  would  be  a  great  omission 
not  to  describe  Koch's  method  in  full. 

Apparatus. — Half  a  dozen  glass  plates,  about  6  by  4 
inches  in  size,  free  from  bubbles  and  scratches  and 
ground  at  the  edges,  are  carefully  cleaned,  placed  in  a 
sheet-iron  box  made  to  receive  them,  and  then  put  in 

the  hot-air  closet,  where 
they  are  sterilized.  The  box, 
which  is  tightly  closed,  al- 
lows the  sterilized  plates  to 
be  kept  on  hand  indefinitely 
before  using. 

A  moist  chamber,  or  double 
dish,  about  10  inches  in  di- 
ameter and  3  inches  deep,  the 
upper  half  being  just  enough 
larger  than  the  lower  to  allow 
it  to  close  over  it,  is  carefully 
washed.  A  sheet  of  bibulous 

paper  is  placed  in  the  bottom,  so  that  some  moisture  can 
be  retained,  and  a  i  :  1000  bichlorid  solution  is  poured  in 
and  brought  in  contact  with  the  sides,  top,  and  bottom 


taught  by  Koch. 


CULTURES,   AND    THEIR  STUDY.  141 

by  turning  the  dish  in  all  directions.  The  solution  is 
emptied  out,  and  the  dish,  which  is  always  kept  closed, 
is  ready  for  use. 

A  levelling  apparatus  is  required  (Fig.  19).  This  con- 
sists of  a  wooden  tripod  with  adjustable  screws,  and  a  glass 
dish  covered  by  a  flat  plate  of  glass  upon  which  a  low 
bell-jar  stands.  The  glass  dish  is  filled  with  broken  ice 
and  water,  covered  with  the  glass  plate,  and  then  exactly 
levelled  by  adjusting  the  screws  under  the  legs  of  the 
tripod.  When  level  the  cover  is  placed  upon  it,  and  it 
is  ready  for  use. 

Method  (Fig.  24). — A  sterile  platinum  loop  is  dipped 
into  the  material  to  be  examined,  a  small  quantity  se- 


FIG.  24. — Method  of  holding  tubes  during  inoculation. 

cured,  and  stirred  about  so  as  to  distribute  it  evenly 
through  a  tube  of  the  melted  gelatin.  If  the  material 
under  examination  is  very  rich  in  bacteria,  one  loopful 
may  contain  a  million  individuals,  which,  if  spread  out 
in  a  thin  layer,  would  develop  so  many  colonies  that  it 
would  be  impossible  to  see  any  one  clearly ;  hence  the 
necessity  for  a  dilution.  From  the  first  tube  a  loopful 
of  gelatin  is  carried  to  a  second  tube  of  melted  gelatin 
and  stirred  well,  so  as  to  distribute  the  organisms  evenly 
through  it.  In  this  tube  we  may  have  no  more  than  ten 
thousand  organisms,  and  if  the  same  method  of  dilution 
be  used  again,  the  third  tube  may  have  only  a  few  hun- 
dreds, and  a  fourth  only  a  few  dozen  colonies. 

After  the  tubes  are  prepared,  one  of  the  sterile  glass 
plates  is  caught  by  its  edges,  removed  from  the  iron  box, 
and  placed  beneath  the  bell-glass  upon  the  cold  plate 


142  PATHOGENIC  BACTERIA. 

covering  the  ice- water  of  the  levelling  apparatus.  The 
plug  of  cotton  closing  the  mouth  of  tube  No.  i  is  re- 
moved, and  to  prevent  contamination  during  the  outflow 
of  the  gelatin  the  mouth  of  the  tube  is  held  in  the  flame 
of  a  Bunsen  burner  for  a  moment  or  two.  The  gelatin 
is  then  cautiously  poured  out  upon  the  plate,  the  mouth 
of  the  tube,  as  well  as  the  plate,  being  covered  by  the 
bell-glass  to  prevent  contamination  by  germs  in  the  air. 
The  apparatus  being  level,  the  gelatin  spreads  out  in  an 
even,  thin  layer,  and,  the  plate  being  cold  from  the  ice 

beneath,  it  immediately  solidi- 
fies, and  in  a  few  moments  can 
be  removed  to  the  moist  cham- 
7^       ber  prepared  to  receive  it.     As 

FIG.  25.— Glass  bench. 

soon  as  plate  No.  i  is  prepared, 

the  contents  of  tube  No.  2  are  poured  upon  plate  No.  2, 
allowed  to  spread  out  and  solidify,  and  then  superimposed 
on  plate  No.  i  in  the  moist  chamber,  being  separated  from 
the  plate  already  in  the  chamber  by  small  glass  benches 
(Fig.  25)  made  for  the  purpose  and  sterilized.  After  the 
contents  of  all  the  tubes  are  thus  distributed,  the  moist 
chamber  and  its  contents  are  allowed  to  stand  for  some 
hours,  to  permit  the  bacteria  to  grow.  Where  each  or- 
ganism falls  a  colony  develops,  and  the  success  of  the 
whole  method  depends  upon  the  isolation  of  a  colony 
and  its  transfer  to  a  tube  of  culture-medium  where  it 
can  grow  unmixed  and  undisturbed. 

The  description  must  have  made  evident  the  fact  that 
only  such  culture-media  can  be  used  for  plate-cultures  as 
can  be  melted  and  solidified  at  will — viz.  gelatin,  agar- 
agar,  and  glycerin  agar-agar.  Blood-serum  and  L,6frler's 
mixture  are  entirely  inappropriate. 

The  great  drawback  to  this  excellent  method  is  the 
cumbersome  apparatus  required  and  the  comparative  im- 
possibility of  making  plate-cultures,  as  is  often  desirable, 
in  the  clinic,  at  the  bedside,  or  elsewhere  than  in  the 
laboratory.  The  method  therefore  soon  underwent  mod- 
ifications, the  most  important  being 


CULTURES,   AND    THEIR  STUDY.  143 

Petri's  Dishes. — These  small  dishes  (Fig.  26),  about 
4  inches  in  diameter  and  j£  inch  deep,  with  accurately 
fitting  lids,  are  about  as  convenient  as  anything  that  has 
been  devised  in  bacteriological  technique.  They  dis- 


FlG.  26. — Petri  dish  for  making  plate-cultures. 

pense  with  plates  and  plate-boxes,  with  moist  chambers 
and  benches,  and  usually  with  the  levelling  apparatus, 
though  this  is  still  employed  in  connection  with  the 
Petri  dishes  in  some  laboratories. 

The  method  of  the  employment  of  Petri  dishes  is  very 
simple.  The  dishes  are  carefully  cleaned,  polished,  and 
sterilized  by  hot  air,  care  being  taken  that  they  are  placed 
in  the  hot-air  closet  right  side  up,  and  after  sterilization 
are  kept  covered  and  in  that  position.  The  dilution  of 
the  material  under  examination  is  made  with  gelatin  or 
agar-agar  tubes  in  the  manner  described  above,  the  plugs 
are  removed,  the  mouth  of  the  tube  is  cautiously  held 
for  a  moment  in  the  flame,  then  the  contents  of  each 
tube  are  poured  into  one  of  the  sterile  dishes,  whose  top 
is  elevated  just  sufficiently  to  allow  the  mouth  of  the 
tube  to  enter.  The  gelatin  is  spread  over  the  bottom 
of  the  dish  in  an  even  layer,  is  allowed  to  solidify, 
labelled,  and  then  stood  away  for  the  colonies  to  develop. 

Esmarch  Tubes. — This  method,  devised  by  Esmarch, 
converts  the  walls  of  the  test-tube  into  the  plate  and  dis- 
penses with  all  other  apparatus.  The  tubes,  which  are 
inoculated  and  in  which  the  dilutions  are  made,  should 
contain  less  than  half  the  usual  amount  of  gelatin  or 
agar-agar.  After  inoculation  the  cotton  plugs  are  pushed 
into  the  tubes  until  even  with  their  mouths,  and  then 
covered  with  a  rubber  cap,  which  protects  them  from 
wetting.  A  groove  is  next  cut  in  a  block  of  ice,  and 


I44  PATHOGENIC  BACTERIA. 

the  tube,  held  almost  horizontally,  is  rolled  in  this  until 
the  entire  surface  of  the  glass  is  covered  with  a  thin 
layer  of  the  solid  medium  (Fig.  27).  Thus  the  tube 
becomes  the  plate  upon  which  the  colonies  develop. 


FIG.  27. — Esmarch  tube  on  block  of  ice  (redrawn  after  Abbott). 

Several  little  points  need  to  be  observed  in  .carrying 
out  Esmarch' s  method.  The  tube  must  not  contain  too 
much  culture-medium,  or  it  cannot  be  rolled  into  an  even 
layer.  In  rolling  the  contents  should  not  touch  the  cotton 
plug,  lest  it  be  glued  to  the  glass  and  its  subsequent  use- 
fulness be  injured.  No  water  must  be  admitted  from  the 
melted  ice. 

The  offspririg  of  each  bacterium  growing  upon  the 
film  of  gelatin  constituting  a  plate-culture  form  a  mass 
which  has  already  been  pointed  out  as  a  colony.  These 
small  bacterial  families  may  be  seen  through  a  micro- 
scope when  still  much  too  small  for  detection  by  the 
naked  eye,  and  because  of  their  minuteness  should  always 
be  studied  with  the  microscope. 

The  original  plates  of  Koch  are  very  inconvenient  for 
such  examination,  because  it  is  impossible  to  remove 
them  from  the  moist  chamber  and  lay  them  upon  the 
stage  of  the  microscope  without  exposing  them  to  the 
danger  of  contamination  by  the  atmosphere,  so  that  the 
advantages  of  Petri  dishes  and  Esmarch  tubes,  where 
the  examination  may  be  made  through  the  glass  tube  or 


CULTURES,   AND    THEIR  STUDY.  145 

through  the  bottom  of  the  inverted  dish,  will  be  more 
than  ever  apparent. 

The  colonies  should  be  viewed  from  time  to  time  in 
their  growth,  drawings  being  made  of  the  appearances, 
so  as  to  form  a  series  showing  the  developmental  cycle. 
Most  colonies  will  be  found  to  originate  as  spherical,  cir- 
cumscribed, slightly  granular,  yellowish,  greenish,  or 
brownish  dots,  and  later  to  send  out  offshoots  or  filaments 
or  to  develop  concentric  rings  or  characteristic  liquefac- 
tions. A  few  appear  from  the  very  first  as  woolly  clumps 
of  entangled  threads. 

Some  of  the  most  diverse  forms  of  colonies  are  repre- 
sented in  the  accompanying  illustrations  (Figs.  28-32). 


FIGS.  28,  29,  30. — The  various  appearances  of  colonies  of  bacteria  under  the 
microscope :  a,  colony  of  Bacillus  liquefaciens  parvus  (Liideritz) ;  b,  colony 
of  Bacillus  polypiformis  (Liborius);  c,  colony  of  Bacillus  radiatus  (Liideritz). 

A  pure  culture,  when  obtained  from  colonies  growing 
upon  a  plate,  must  always  be  made  from  a  single  colony, 
the  transplantation  being  accomplished  under  a  low  power 
of  the  microscope.  The  naked  eye  can  rarely  be  depended 
upon  to  recognize  the  purity  of  a  colony  or  its  isolation. 

Selecting  as  isolated,  large,  and  characteristic  a  colony 
as  possible,  it  is  brought  to  the  centre  of  the  field.  A 
platinum  wire,  securely  fused  into  a  glass  handle  about 
8  inches  long,  is  sterilized  by  being  made  incandescent 
in  a  Bunsen  flame,  cooled,  and  then  cautiously  manipu- 
lated until,  while  it  is  watched  through  the  microscope, 
10 


146  PATHOGENIC  BACTERIA. 

it  is  seen  to  touch  the  colony  and  take  part  of  its  con- 
tents away.  /;/  this  maneuvre  the  wire  must  not  touch 
the  objective,  the  glass,  or  anything  except  the  colony. 
Having  secured  the  adhesion  of  a  few  bacteria  to  the 
sterile  wire,  the  pure  culture  is  made  by  introducing 
them  into  a  sterile  culture-medium. 

If  the  pure  culture  is  to  be  made  in  bouillon,  the  tube 
is  held  obliquely,  so  that  when  the  cotton  plug  is  cau- 
tiously removed  no  germs  can  fall  in  from  the  air.  The 
plug  is  removed  by  a  twisting  movement.  The  wire,  with- 
out being  allowed  to  touch  the  mouth  or  sides  of  the 
tube,  is  plunged  into  its 
contents  and  stirred  about 
until  the  bacteria  are  de- 
tached, and  is  then  re- 


FIGS.  31,  32. — The  various  appearances  of  colonies  of  bacteria  under  the 
microscope  :  a,  colony  of  Bacillus  muscoides  (Liborius)  ;  b,  colony  of  Bacillus 
nnthrnci*  (FlUgge). 

moved  and  the  plug  replaced.  The  wire  should  be  im- 
mediately sterilized  by  heating  to  incandescence,  lest  the 
bacteria  be  pathogenic  and  capable  of  doing  subsequent 
harm. 

If  the  culture  is  to  be  made  in  gelatin,  a  different 
method  is  employed.  The  tube  is  either  held  horizon- 
tally, or,  as  is  perhaps  better,  inverted  ;  the  cotton  plug 


CULTURES,   AND    THEIR  STUDY.  147 

is  removed  cautiously  ;  the  wire  bearing  the  bacteria 
from  the  colony  is  introduced  until  its  point  enters  the 
centre  of  the  gelatin,  and  is  then  carefully  pushed  on 
until  a  vertical  puncture  from  the  surface  to  the  bottom 
of  the  gelatin  is  made.  This  is  the  puncture-culture — 
u  stichcultur  "  of  the  Germans. 

If  the  bacteria  are  only  to  be  planted  upon  the  surface 
of  the  culture-medium,  the  wire  is  drawm  over  the  surface 
of  a  tube  of  obliquely  solidified  gelatin,  agar-agar,  blood- 
serum,  etc.  with  a  steady,  slow  movement,  so  as  to  scatter 
the  germs  along  its  path  and  cause  the  development  of 
the  bacteria  in  an  enormous  colony  or  mass  of  colonies 
in  a  line  following  the  longest  diameter  of  the  exposed 
surface  from  end  to  end.  This  is  the  stroke-culture — 
"strichcultur." 

The  method  of  holding  the  tubes,  cotton  plugs,  and 
platinum  wire  during  the  process  of  inoculation  is  shown 
in  Figure  20. 

Sometimes  it  is  desirable  to  preserve  an  entire  colored 
colony  as  a  microscopic  specimen.  To  do  this  a  perfectly 
clean  cover-glass,  not  too  large  in  size,  is  momentarily 
warmed,  then  carefully  laid  upon  the  surface  of  the 
gelatin  or  agar-agar  containing  the  colonies.  Sufficient 
pressure  is  applied  to  the  surface  of  the  glass  to  exclude 
bubbles  underneath,  but  the  pressure  must  not  be  too 
great,  as  it  may  destroy  the  integrity  of  the  colony. 
The  cover  is  gently  raised  by  one  edge,  and  if  successful 
the  whole  colony  or  a  number  of  colonies,  as  the  case 
may  be,  will  be  found  adhering  to  it.  It  is  treated 
exactly  as  any  other  cover-glass  preparation,  is  dried, 
fixed,  stained,  and  mounted,  and  kept  as  a  permanent 
specimen.  It  is  called  an  adhesion  preparation — u  klatsch 
praparat. ' ' 

Very  often,  when  one  is  in  a  hurry,  pure  cultures  from 
single  colonies  may  be  secured  by  a  very  simple  manipu- 
lation suggested  by  Banti.1  The  inoculation  is  made 
into  the  water  of  condensation  at  the  bottom  of  an  agar- 

1  Centralbl.  f.  Bakt.  und  Parasitenk.,  1895,  xvii.,  No.  16. 


148  PATHOGENIC  BACTERIA. 

agar  tube,  without  touching  the  surface.  The  tube  is 
then  inclined  so  that  the  water  flows  over  the  agar, 
after  which  it  is  stood  away  in  the  vertical  position. 
Colonies  will  grow  where  bacteria  have  been  floated  upon 
the  agar-agar,  and  may  be  picked  up  later  in  the  same 
manner  as  from  a  plate. 

In  other  cases  pure  cultures  may  best  be  secured  by 
animal  inoculation.  For  example,  when  the  tubercle 
bacillus  is  to  be  isolated  from  milk  or  urine  which  con- 
tains rapidly  growing  bacteria  that  would  outgrow  the 
slow-developing  tubercle  bacillus,  it  is  better  to  inject 
some  of  the  fluid  into  the  abdominal  cavity  of  a  guinea- 
pig  and  await  the  development  of  tuberculosis,  and  then 
seek  to  secure  the  bacillus  from  the  unmixed  material  in 
the  softened  lymphatic  glands.  Anthrax  bacilli  are  also 
more  easily  secured  in  pure  culture  by  inoculating  a 
mouse  and  recovering  the  bacilli  from  a  spleen  or  the 
heart's  blood  after  death,  than  by  going  to  the  trouble  of 
making  plates  and  picking  out  the  colonies. 

In  many  cases  when  it  is  desired  to  isolate  the  micro- 
coccus  tetragenus,  the  pneumococcus,  and  others,  it  is 
easier  to  inoculate  the  most  susceptible  animal  and 
recover  the  germ  from  the  organs  than  to  plate  it  out  and 
search  for  the  colony  among  many  others  which  may  be 
similar  to  it. 

The  development  of  bacteria  in  liquids  is  of  less  in- 
terest than  that  upon  solid  media.  The  growth  generally 
manifests  itself  by  a  diffused  turbidity.  Sometimes  flocculi 
float  in  the  otherwise  clear  medium.  Some  forms  grow 
most  rapidly  at  the  surface  of  the  liquid,  and  produce  a 
distinct  membranous  pellicle  called  a  mycoderma.  In 
such  a  growth  multitudes  of  degenerated  bacteria  and 
large  numbers  of  spores  are  to  be  observed.  On  the 
other  hand,  it  occasionally  happens  that  the  growth 
occurs  chiefly  below  the  surface,  and  may  produce  gelat- 
inous masses  which  are  known  as  sooglea. 

In  gelatin  the  bacteria  exhibit  a  great  variety  of  ap- 
pearances, many  of  which  are  beautiful  and  interesting. 


CULTURES,  AND    THEIR  STUDY. 


149 


Certain  bacteria,  as  the  tubercle  bacillus,  will  not  grow 
at  all  upon  gelatin.  Some  forms  which  are  rigidly  ae- 
robic will  only  grow  upon  or  near  the  surface  ;  others, 
anaerobic,  only  in  the  deeper  parts.  The  majority,  how- 
ever, grow  both  upon  the  surface  and  in  the  puncture 
made  by  the  wire.  Sometimes  the  consistence  of  the 
gelatin  is  unaltered  ;  sometimes  it  is  liquefied  throughout, 
sometimes  only  at  the  surface.  Sometimes  offshoots  ex- 
tend from  the  colonies  into  the  gelatin,  giving  the  culture 


FIG.  33. — Various  forms  of  gelatin  puncture-cultures :  a,  Bacillus  typhi  ab- 
dominalis;  b,  B.  anthracis;  c,  B.  mycoides;  d,  B.  mesentericus  vulgatus; 
e,  B.  of  malignant  edema ;  f,  B.  radiatis. 

a  bristling  appearance.  Figure  33  will  serve  to  illustrate 
different  varieties  of  gelatin  growth. 

The  growth  in  gelatin  is  generally  so  far  removed  from 
the  walls  of  the  tube  (a  central  puncture  nearly  always 
being  made  in  the  culture-medium,  in  order  that  the 
growth  be  symmetrical)  that  it  is  next  to  impossible  to 
make  a  microscopical  examination  of  it  with  any  power 
beyond  that  given  by  a  hand-lens. 

Much  attention  has  been  given  of  late  to  the  preparation 
of  microtome  sections  of  the  gelatin  growth.  To  accom- 
plish this  the  glass  is  warmed  sufficiently  to  allow  the 
gelatin  to  be  removed  and  placed  in  Miiller's  fluid  (bi- 


150  PATHOGENIC  BACTERIA. 

chromate  of  potassium  2. -2. 5,  sulphate  of  sodium  i, 
water  100),  wliere  it  is  hardened.  When  quite  firm  it 
is  washed  in  water,  passed  through  alcohols  ascending 
in  strength  from  50  to  100  per  cent.,  imbedded  in  cel- 
loidin,  cut  wet,  and  stained  like  a  section  of  tissue. 

A  ready  method  of  doing  this  has  been  suggested  by 
Winkler,1  who  bores  a  hole  in  a  block  of  paraffin  with 
the  smallest-size  cork-borer,  soaks  the  block  in  bichlorid 
solution  for  an  hour,  pours  liquid  gelatin  into  the  cavity, 
allows  it  to  solidify,  inoculates  it  by  the  customary  punc- 
ture of  the  platinum  wire,  allows  it  to  develop  sufficiently, 
and  when  ready  cuts  the  sections  under  alcohol,  subse- 
quently staining  them  with  much-diluted  carbol-fuchsin. 

Very  pretty  museum  specimens  of  plate-  and  puncture- 
cultures  in  gelatin  can  be  made  by  simultaneously  killing 
the  micro-organisms  and  permanently  fixing  the  gelatin 
with  formalin,  which  can  either  be  sprayed  upon  the 
gelatin  or  applied  in  dilute  solution.  As  gelatin  fixed 
in  formalin  cannot  subsequently  be  liquefied,  such  prep- 
arations will  last  indefinitely. 

The  growths  which  occur  upon  agar-agar  are  in  many 
ways  less  characteristic  than  those  in  gelatin,  but  as  this 
medium  does  not  liquefy  except  at  a  high  temperature 
(100°  C.),  it  has  that  great  advantage  over  gelatin.  The 
colorless  or  almost  colorless  condition  of  the  preparation 
also  aids  in  the  detection  of  such  chromogenesis  as  may 
be  the  result  of  the  micro-organismal  growth. 

Sometimes  the  growth  is  colored,  sometimes  not ;  some- 
times the  production  of  a  soluble  pigment  colors  the 
agar-agar  as  well  as  the  growth  ;  sometimes  the  growth 
is  one  color  and  the  agar-agar  another.  Sometimes  the 
growth  is  filamentous,  sometimes  a  smooth,  shining  band. 
Occasionally  the  bacterium  does  not  grow  upon  agar-agar 
unless  glycerin  be  added  (tubercle  bacillus) ;  sometimes 
it  will  not  grow  even  then  (gonococcus). 

Still  less  characteristic  are  the  growths  upon  potato. 
Most  bacteria  produce  rather  smooth,  shining,  irregu- 

1  Fortschritte  der  .]/,•,//«•/«,  Bd.  xi.,  1893,  No-  22. 


CULTURES,  AND    THEIR  STUDY.  151 

larly-extending  growths,  which  often  show  very  beautiful 
colors. 


FIG.  34. — New  model  incubating-oven  with  electro-regulator. 

Ill  milk  and  litmus  milk  one  must  observe  the  presence 
or  absence  of  acid-production,  the  coagulation  which  may 


152  PATHOGENIC  BACTERIA. 

or  may  not  accompany  it,  and  the  subsequent  gelatiniza- 
tion  or  digestion  of  the  coagulum. 

Blood-serum  is  liquefied  by  some  bacteria.  The  ma- 
jority of  organisms  are  not  very  characteristic  in  their 
development  upon  it.  Others,  as  the  bacillus  of  diph- 
theria, are,  however,  characterized  by  their  shape,  color, 
and  rapidity  of  development  at  given  temperatures. 

While  most  of  the  saprophytic  bacteria  will  grow  well 
at  the  ordinary  temperature  of  a  well-warmed  room,  the 
important  pathogenic  forms  require  to  be  kept  at  the 
temperature  of  the  body.  To  do  this  accurately  an  in- 
cubating oven  becomes  a  necessity.  Various  forms,  of 
wood  and  metal,  are  in  the  market,  the  one  shown  in  the 
illustration  (Fig.  34)  being  one  of  the  newest  and  best. 

It  scarcely  need  be  pointed  out  that  gelatin  cultures 
cannot  be  grown  in  the  incubating  oven,  as  the  medium 
will  not  remain  solid  at  temperatures  above  20-22°  C. 


CHAPTER    VIII. 
THE   CULTIVATION  OF  ANAEROBIC  BACTERIA. 

THE  cultivation  of  micro-organisms  which  will  not 
grow  where  the  least  amount  of  oxygen  is  present  is 
always  attended  with  much  difficulty,  and  can  seldom  be 
accomplished  with  certainty.  Many  methods  have  been 
suggested,  but  not  one  can  be  described  as  satisfactory. 

Koch  originally  cultivated  anaerobic  bacteria  upon 
plates  by  covering  the  surface  of  the  soft  gelatin  with  a 
thin  film  of  mica  previously  sterilized  by  incandescence. 
Some  anaerobic  forms  will  grow  quite  well  by  such  a 
simple  exclusion  of  the  air,  but  the  strictly  anaerobic 
forms  will  not  develop  at  all. 

Hesse  originated  the  plan,  still  sometimes  followed,  of 
making  a  deep  puncture  in  recently  boiled  and  rapidly 
sterilized  gelatin  or  agar-agar,  then  covering  the  surface 
with  sterilized  oil,  through  which  no  oxygen  was  sup- 
posed to  penetrate  (Fig.  35). 

Liborius  suggested  the  plan  of  having  a  tube  nearly 
full  of  gelatin  or  agar-agar,  boiling  it  just  before  inocu- 
lation, so  as  to  expand  and  drive  out  whatever  air  it 
might  contain,  making  the  inoculation  while  the  culture- 
medium  was  still  fluid,  cooling  rapidly  in  ice-water,  and 
sealing  up  the  tube  in  a  blowpipe  as  near  the  surface  of 
the  gelatin  as  possible. 

Esmarch  used  a  regular  "  Esmarch  tube,"  into  the 
central  cavity  of  which  melted  sterile  gelatin  was  poured 
to  exclude  the  air. 

Buchner  invented  a  method  by  which,  by  the  use  of 
pyrogallic  acid,  the  oxygen  was  absorbed  from  the  atmo- 
sphere in  which  the  culture  was  kept,  and  the  growth 
allowed  to  continue  in  the  nitrogen  and  carbonic  acid 

153 


154  PATHOGENIC  BACTERIA. 

which  remained  (Fig.  36).  His  method  was  to  place  the 
tube  which  had  been  inoculated  in  a  much  larger  outer 
test-tube  containing  alkaline  pyrogallic  acid.  The  large 


FIG.  35.  — Hesse's 
method  of  making 
anaerobic  cultures. 


FIG.  36.— Buchner's 
method  of  making  an- 
aerobic cultures. 


FIG.  37. — Frankel's  meth- 
od of  making  anaerobic  cul- 
tures. 


tube  was  closed  with  a  rubber  cap,  and  the  absorption  of 
the  oxygen  allowed  to  progress. 

Gruber,  instead  of  absorbing  the  oxygen  as  Buchner 
does,  prefers  to  use  an  air-pump  and  exhaust  the  contents 
of  the  tube.  He  uses  a  tube  having  a  slender  neck  and 
a  perforated  rubber  stopper.  After  the  inoculation  is 
made  the  air  is  pumped  out  and  the  slender  neck  sealed 
in  tlu-  blowpipe.  After  this  the  tube  can  be  warmed  and 
the  melted  gelatin  or  agar-agar  rolled  on  its  sides,  as  sug- 
gested by  Esmarch,  if  desired. 

Better  than  any  of  the  preceding  is  the  method  of 
Frinki-1,  which  removes  the  air  and  replaces  it  by  hy- 
drogen. Frankel  prepares  an  ordinary  Esmarch  tube, 
removes  the  cotton  stopper,  and  replaces  it  by  a  carefully 
sterilized  rubber  cork  containing  two  tubes  (Fig.  37).  The 


CULTIVATION  OF  ANAEROBIC  BACTERIA.    155 

tubes  are  connected  with  a  hydrogen  generator,  and  the 
gas  is  allowed  to  pass  through  until  all  the  oxygen  is 
forced  out  and  replaced  by  the  hydrogen,  after  which  the 
ends  of  the  tubes  are  sealed  in  the  flame  (Fig.  36). 

Iviborius  has  designed  a  special  tube  for  accomplish- 
ing the  same  thing. 

Kitasato  and  Weil  found  the  addition  of  0.3-0.5  per 
cent,  of  sodium  formate  to  be  of  use  in  aiding  the  rapid- 
ity of  the  development  of  anaerobic  cultures.  Iviborius 
found  that  2  per  cent,  of  glucose  added  to  the  culture- 
medium  also  increased  the  rapidity  of  the  process. 

The  methods  now  generally  employed  by  bacteri- 
ologists for  the  anaerobic  cultivations  embrace  all  the 
essentials  of  the  foregoing  methods.  One  of  the  best 
arrangements  for  the  purpose  is  that  devised  by  Dr. 
Ravenel.  His  inoculations  are  deeply  made  in  culture- 
media  as  free  from  air  as  possible.  The  tubes  are 
loosely  plugged,  and  are  placed  in  an  air-tight  cham- 
ber the  bottom  of  which  contains  pyrogallic  acid — py- 
rogallic  acid  i,  solution  of  caustic  potash  i,  water  10. 
The  apparatus  is  connected  by  two  tubes  with  an  ex- 
haust-pump on  one  side,  and  with  a  hydrogen  appara- 
tus on  the  other,  by  which  means  the  atmosphere  is  ex- 
hausted, and  replaced  by  hydrogen  until  only  pure  hydro- 
gen remains,  after  which  the  chamber  is  permanently 
sealed  and  the  germs  allowed  to  grow.  Such  a  chamber 
can  be  constructed  to  hold  a  number  of  tubes  or  Petri 
dishes,  yet  not  be  too  large  to  be  stood  in  an  incubator. 
Whatever  oxygen  may  have  escaped  the  exhaustion  or 
have  entered  by  the  process  of  leakage  is  at  once  absorbed 
by  the  pyrogallic  acid  in  the  lower  chamber  of  the  ap- 
paratus. 

Apparatus  for  plating  out  strictly  anaerobic  bacteria 
that  have  met  with  great  favor  are  those  invented  by 
Botkin  (Fig.  38)  and  Novy  (Fig.  39).  The  first  mentioned 
combines  the  replacement  of  the  air  by  hydrogen  and  the 
absorption  of  the  oxygen  possibly  remaining  by  alkaline 
pyrogallic  acid;  the  other  simply  replaces  the  oxygen  by 


156 


PATHOGENIC  BACTERIA. 


hydrogen.  In  using  Botkin's  apparatus  the  uncovered 
Petri  dishes  are  placed  one  above  the  other  in  the  rack  c, 
and  covered  with  the  bell-glass  A.  Liquid  paraffin  is 
poured  in  the  dish  B  until  it  is  about  half  full.  From  a 

Kipp's  apparatus  hydrogen 
gas  enters  the  little  rubber 
tube  <z,  subsequently  escap- 
ing by  the  tube  b.  When 
only  pure  hydrogen  escapes 
the  rubber  tubes  a  and  b  are 
withdrawn,  and  the  appa- 
ratus remains  filled  with  hy- 
drogen. Lest  a  little  oxygen 
should  remain,  it  is  best  to 
have  the  dishes  at  the  top 
and  bottom  of  the  rack  filled 
with  alkaline  pyrogallic 
acid.  Tetanus  can  be  cul- 
tivated in  this  apparatus. 
The  jars  recently  intro- 

j          j    v       XT  *1 

duced  bv  Now  are  similar 


FlG.  38.  —  Botkin's  apparatus  for  male- 
ing  anaerobic  cultures. 


in      principle,      depending 
upon  the  replacement  of  the  air  by  hydrogen.     They  are 


in;     }Q. — Novy's  jars  for  anaerobic 


so  constructed  that  when  the  stopper  occupies  a  certain 
relative  position  to  the  neck  the  gas  can  enter  and  exit, 


CULTIVATION  OF  ANAEROBIC  BACTERIA.     157 

but  when  the  stopper  is  turned  a  little  the  jar  is  hermet- 
ically sealed.  Alkaline  pyrogallic  acid  in  a  test-tube,  or 
in  the  bottom  of  the  jar,  will  serve  to  absorb  any  remain- 
ing oxygen.  The  larger  jar  (Fig.  39,  a)  is  intended  for 
Petri  dishes,  the  smaller  one  (b)  for  test-tube  cultures. 
Roux  has  suggested  the  simplest  method  of  cultivating 
anaerobic  bacteria.  The  germs  are  distributed  through 
freshly  boiled,  still  liquid,  gelatin  or  agar-agar,  as  in 
making  the  dilutions  for  plate-cultures,  then  drawn  into 
a  long,  slender  sterile  piece  of  glass  tubing  of  small 
calibre.  When  the  tube  is  full  the  ends,  which  should 
have  been  narrowed,  are  closed  in  a  flame,  and  the  cul- 
ture is  hermetically  sealed  in  an  air-tight  chamber.  The 
chief  difficulty  is  in  transplanting  the  growing  colony. 
To  do  this  the  tube  must  be  opened  with  a  file  or  a  dia- 
mond at  the  point  where  the  colony  desired  is  observed. 


CHAPTER   IX. 
EXPERIMENTATION  UPON  ANIMALS. 

BACTERIOLOGY  has  to-day  become  a  science  whose 
principal  objects  are  to  discover  the  cause,  explain  the 
symptoms,  and  prepare  the  cure  of  diseases.  We  can- 
not hope  to  achieve  these  objects  except  by  the  intro- 
duction of  bacteria  into  animals,  where  their  effects  and 
the  effects  of  their  products  can  be  studied. 

No  one  should  more  heartily  condemn  wanton  cruelty 
to  animals  than  the  physician  and  the  naturalist.  In- 
deed, it  is  hard  to  imagine  a  class  of  men  so  much  of 
whose  lives  is  spent  in  relieving  pain,  and  who  know  so 
much  about  pain,  being  guilty  of  the  wholesale  butchery 
and  torture  accredited  to  them  by  a  few  of  the  laity, 
whose  eyes,  but  not  whose  brains,  have  looked  over 
the  pages  of  physiological  text-books. 

Experimentation  upon  animals  has  given  us  almost 
all  our  knowledge  of  physiology,  most  of  our  valuable 
therapeutics,  and  the  only  scientific  methods  of  treating 
tetanus  and  diphtheria. 

Experiments  upon  animals  we  must  make,  and,  as 
animals  differ  in  their  susceptibility  to  diseases,  large 
numbers  and  different  kinds  must  be  employed. 

The  bacteriological  methods  are  not  cruel.  Two  prin- 
cipal modes  of  introducing  bacteria  are  employed:  the 
su1>rutuneotis  injection  and  the  intravenous  injection. 

Subcutaneous  injections  into  animals  are  made  exactly 
as  hypodermic  injections  are  given  to  man. 

Any  hypodermic  syringe  that  can  be  conveniently 
cleaned  and  disinfected  may  be  employed  for  the  purpose. 
Those  expressly  designed  for  bacteriological  work  and 
most  frequently  employed  are  shown  in  Fig.  40.  Those 

158 


EXPERIMENTA  TION  UPON  ANIMALS. 


159 


of  Meyer  and  Roux  resemble  ordinary  hypodermic 
syringes;  that  of  Koch  is  supposed  to  possess  the  decided 
advantage  of  not  having  a  piston  to  come  into  contact 
with  the  fluid  to  be  injected.  This  is,  however,  some- 
what disadvantageous  inasmuch  as  the  cushion  of  com- 
pressed air  that  drives  out  the  contents  is  elastic,  and  un- 
less carefully  watched  will  follow  the  injection  into  the 
body  of  the  animal.  In  making  subcutaneous  injections 
there  is  no  disadvantage  or  danger  from  the  entrance  of 


FIG.  40.— I,  Roux's  bacteriological  syringe;    2,  Koch's  syringe;    3,  Meyer's 
bacteriological  syringe. 

air  beneath  the  skin,  but  in  intravenous  injections  it  is 
commonly  supposed  to  be  dangerous. 

All  syringes  should  be  disinfected  with  carbolic  acid 
solutions  before  and  after  using,  the  carbolic  acid  being 
allowed  to  act  for  some  time  and  then  washed  out 
with  sterile  water.  Syringes  should  not  be  boiled,  as 
it  ruins  the  packings,  whether  of  asbestos,  leather,  or 
rubber. 

The  intravenous  injections  differ  only  in  that  the  needle 
of  the  syringe  is  introduced  into  a  vein.  This  is  easy  in  a 
large  animal  like  a  horse,  but  is  very  difficult  in  a  small 
animal,  and  wellnigh  impossible  in  anything  smaller  than 
a  rabbit.  Such  injections  when  given  to  rabbits  are  gen- 
erally made  into  the  ear-veins,  as  those  most  conspicuous 
and  accessible  (Fig.  41).  A  peculiar  and  important  fact 
to  remember  is,  that  the  less  conspicuous  posterior  vein 


i6o 


PATHOGENIC  BACTERIA. 


is  much  better  adapted  to  the  purpose  than  the  anterior. 

The  introduction  of  the  needle  should  be  made  from  the 

hairy   surface   of  the 
ear. 

If  the  ear  is  manip- 
ulated for  a  moment  or 
two  before  the  injec- 
tion is  begun,  vaso- 
motor  dilatation 
occurs  and  the  blood- 
vessels all  become 
larger  and  more  con- 
spicuous. The  vein 
should  be  compressed 
at  the  root  of  the  ear 
until  the  needle  is  in- 
troduced, and  the  in- 

FIG.  41. — Method  of  making  an  intravenous    jection  made   as   near 
injection  into  a  rabbit.    Observe  that  the  needle   ^e  rooj-  as  possible 
enters  the  posterior  vein  from  the  hairy  surface.  .  - 

The  introduction  of 

bacteria  into  the  lymphatics  is  only  possible  by  injecting 
liquid  preparations  of  them  into  some  organ  with  com- 
paratively few  blood-vessels  and  large  numbers  of  lym- 
phatics. The  testicle  is  best  adapted  to  this  purpose,  the 
needle  being  introduced  deeply  into  the  organ. 

Sometimes  the  inoculation  can  be  made  by  the  platinum 
wire,  a  very  small  opening  made  in  the  skin  by  a  snip  of 
the  scissors  being  sufficient. 

Sometimes  intra-abdominal  and  intra-pleural  injections 
are  made,  and  in  cases  where  it  becomes  necessary  to 
determine  the  presence  or  absence  of  tuberculosis  or 
glanders  in  tissues  it  may  be  necessary  to  introduce  small 
pieces  of  the  suspected  tissue  under  the  skin  or  into  the 
abdominal  cavities.  To  do  this  is  not  difficult.  The 
hair  is  carefully,  closely  cut  over  the  point  of  election, 
which  is  generally  on  the  abdomen  near  the  groin,  the 
skin  picked  up  with  forceps,  a  snip  made  through  it, 
and  the  points  of  the  scissors  introduced  for  half  an  inch 


EXPERIMENTATION  UPON  ANIMALS. 


161 


or  so  and  then  separated.  By  this  maneuver  a  subcuta- 
neous pocket  is  formed,  into  which  the  tissue  is  easily 
forced.  The  opening  should  not  be  large  enough  to  re- 
quire subsequent  stitching. 

Small  animals,  like  rabbits  and  guinea-pigs,  can  be  held 
in  the  hand,  as  a  rule.  Rabbit-holders  of  various  forms 
can  be  obtained  from  dealers.  Dogs,  cats,  sheep,  and  goats 
can  be  tied  and  held  in  troughs.  A  convenient  form  of 
mouse-holder,  invented  by  Kitasato,  is  shown  in  Fig.  42. 

In  all  these  experiments  one  must  remember  that  the 
amount  of  material  introduced  into  the  animal  must  be 
in  proportion  to  its  size,  and  that  injection-experiments 
upon  mice  generally  are  so  crude  and  destructive  as  to 
warrant  the  comparison  drawn  by  Frankel,  that  to  inject 
a  few  minims  of  liquid  into  the  pleural  cavity  of  a  mouse 
is  u  much  the  same  as  if  one  would  inject  through  a  fire- 
hose three  or  four  quarts  of  some  liquid  into  the  respira- 
tory organs  of  a  man. ' ' 

The  blood  of  animals,  when  it  is  necessary  to  experi- 
ment with  it,  is  best  secured  from 
a  large  vein,  generally  the  jugu- 
lar. From  small  animals,  such  as 
guinea-pigs,  it  may  be  secured  by 
introducing  a  small  cannula  into 
the  carotid  artery. 

Our  observations  of  animals  by 
no  means  cease  with  their  death. 
Indeed,  he  cannot  be  a  bacteriol- 
ogist who  is  f  not  already  a  good 
pathologist  and  expert  in  the  recog- 
nition of  diseased  organs. 

When  an  autopsy  is  to  be  made 
upon  a  small  animal,  it  is  best  to 
wash   it  for  a  few  moments   in  a 
disinfecting  solution,   to   kill    the 
germs  present  upon  the  hair  and  the  skin,  as  well  as  to 
moisten  the  hair   and   enable  it  to    be  kept  out  of  the 
incision. 
11 


FIG.  42. — Mouse-holder. 


i62  PATHOGENIC  BACTERIA. 

The  animal  should  be  tacked  to  a  board  if  small,  or 
tied,  by  cords  fastened  to  the  legs,  to  the  corners  of  a 
table  if  large,  and  should  be  dissected  with  sterile  knives 
and  scissors.  When  a  culture  is  to  be  made  from  the 
interior  of  an  organ — say  the  spleen — it  should  be  incised 
deeply  with  a  sterile  knife  and  the  culture  made  from 
its  centre. 

Fragments  intended  for  subsequent  microscopical  ex- 
amination should  be  cut  very  small  (cubes  of  i  c.cm.)r 
placed  in  absolute  alcohol  for  a  few  hours,  then  trans- 
ferred to  weaker  alcohol,  80-90  per  cent.,  for  preserva- 
tion. The  technique  of  imbedding  and  staining  the  tis- 
sues can  be  found  in  almost  any  reliable  text-book  on 
pathology  or  on  the  special  subject  of  microscopical 
technique. 


CHAPTER    X. 
THE  RECOGNITION  OF  BACTERIA. 

THE  most  difficult  thing  in  bacteriology  is  to  be  able 
to  recognize  the  bacteria  which  come  under  observation. 

A  certain  few  micro-organisms  are  so  characteristic  in 
shape  and  grouping  as  to  be  separated  by  a  microscopic 
examination.  Some,  as  the  tubercle  bacillus,  are  charac- 
teristic in  their  reaction  to  the  anilin  dyes,  and  can  be 
differentiated  at  once  by  this  peculiarity.  Some,  as  the 
Bacillus  mycoides,  are  so  characteristic  in  their  agar-agar 
growth  as  to  eliminate  others.  The  red  color  of  Bacillus 
prodigiosus  and  the  blue  of  Bacillus  janthinus  will  speak 
almost  positively  for  them.  The  potato  culture  of  the 
Bacillus  mesentericus  fuscus  and  its  close  relative  the  vul- 
gatus  is  quite  sufficient  to  enable  us  to  pronounce  upon 
them.  Unfortunately,  however,  there  are  several  hun- 
dreds of  described  species  which  lack  any  one  distinct 
character  that  may  be  used  for  differential  purposes,  and 
require  that  for  their  diagnosis  we  shall  wellnigh  ex- 
haust the  bacteriological  technique  in  an  almost  fruitless 
effort  to  recognize  them. 

A  series  of  useful  tables  has  been  compiled  by  Eisen- 
berg,  and  is  now  almost  indispensable  to  the  worker. 
Unfortunately,  in  tabulating  bacteria  we  constantly  meet 
species  described  so  insufficiently  as  to  make  them  worse 
than  useless  on  account  of  the  confusion  caused. 

The  only  way  to  recognize  a  species  is  to  study  it 
thoroughly  and  compare  it,  step  by  step,  with  the  descrip- 
tions and  tables  of  known  species  compiled  by  Eisenberg 
and  others. 

163 


CHAPTER  XI. 
THE   BACTERIOLOGIC  EXAMINATION  OF  THE  AIR. 

IT  has  been  repeatedly  emphasized — and  indeed  at  the 
present  time  almost  every  one  knows — that  micro-organ- 
isms float  almost  everywhere  in  the  air,  and  that  their 
presence  there  is  a  constant  source  of  danger,  not  only 
of  contamination  in  our  bacteriologic  researches,  but 
also  a  menace  to  our  health. 

Such  micro-organisms  are  neither  ubiquitous  nor  equally 
disseminated,  but  are  much  more  numerous  where  the  air 
is  dusty  than  where  it  is  pure — much  more  so  where  men 
and  animals  are  accustomed  to  live,  than  upon  the  ocean 
or  upon  high  mountain-tops.  The  purity  of  the  atmo- 
sphere bears  a  distinct  relation  to  the  purity  of  the  soil 
over  which  its  currents  blow. 

The  micro-organisms  that  occur  in  the  air  are  for  the 
most  part  harmless  saprophytes  which  have  been  sepa- 
rated from  their  nutrient  birthplace  and  carried  about  by 
the  wind.  They  are  almost  always  taken  up  from  dried 
materials,  experiment  having  shown  that  they  arise  from 
the  surfaces  of  liquids  in  which  they  grow  with  much  dif- 
ficulty. They  are  by  no  means  all  bacteria,  and  a  plate 
of  sterile  gelatin  exposed  for  a  brief  time  to  the  air  will 
generally  grow  moulds  and  yeasts  as  well  as  bacteria. 

The  bacteria  present  are  occasionally  pathogenic,  espe- 
cially in  localities  where  the  discharges  of  diseased  animals 
have  been  allowed  to  collect  and  dry.  For  this  reason  the 
atmosphere  of  the  wards  of  hospitals  and  of  rooms  in 
which  infectious  cases  are  being  treated  is  much  more 
apt  to  contain  them  than  the  air  of  the  street.  However, 
the  dried  expectoration  of  cases  of  tuberculosis,  of  in- 

164 


BACTERIOLOGIC  EXAMINATION  OF  AIR.       165 

fluenza,  and  sometimes  of  pneumonia,  causes  the  specific 
bacteria  of  these  diseases  to  be  far  from  uncommon  in 
street-dust. 

Giinther  points  out  that  the  majority  of  the  bacteria 
which  occur  in  the  air  are  cocci,  sarcina  being  very 
abundant.  Most  of  them  are  chromogenic  and  do  not 
liquefy  gelatin.  It  is  unusual  to  find  a  considerable 
variety  of  bacteria  at  a  time  ;  generally  not  more  than 
two  or  three  species  are  found. 

It  is  an  easy  matter  to  determine  whether  bacteria  are 
present  in  the  air  or  not,  all  that  is  necessary  being  to 
expose  sterile  plates  or  Petri  dishes  of  gelatin  to  the  air 
for  a  while,  close  them,  and  observe  whether  or  not  bac- 
teria grow  upon  them. 

To  make  a  quantitative  estimation  is,  however,  much 


FIG.  43. — Hesse's  apparatus  for  collecting  bacteria  from  the  air. 

more  difficult.     Several  methods  have  been  suggested,  of 
which  the  most  important  may  be  considered. 

The  method  suggested  by  Hesse  is  simple  and  good. 
It  consists  in  making  a  measured  quantity  of  the  air  to 


i66 


PATHOGENIC  BACTERIA. 


be  examined  pass  through  a  horizontal  sterile  tube  about 
70  cm.  long  and  3.5  cm.  wide  (Fig.  43),  the  interior  of 
which  is  coated  with  gelatin  in  the  same  manner  as  an 
Esmarch  tube.  The  tube,  having  been  prepared,  is 
closed  at  both  ends  with  sterile  corks  carrying  smaller 
glass  tubes  closed  with  cotton.  When  ready  for  use  the 
tube  at  one  end  is  attached  to  a  hand-pump,  the  cotton 
is  removed  from  the  other  end,  and  the  air  passed  through 
very  slowly,  the  bacteria  having  time  to  precipitate  upon 
the  gelatin  as  they  pass.  When  the  required  amount  has 
passed  the  tubes  are  again  plugged,  the  apparatus  stood 
away  for  a  time,  and  subsequently,  when  they  have 
grown,  the  colonies  are  counted.  The  number  of  colo- 
nies in  the  tube  will  represent  pretty  accurately  the 
number  of  bacteria  in  the  amount  of  air  which 
passed  through  the  tube. 

In  such  a  cylindrical  culture  it  will  be  noted 
that  if  the  air  is  passed  through  with  the 
proper  slowness,  the  colonies  will  be  much 
more  numerous  near  the  end  of  entrance  than 
that  of  exit.  The  first  to  fall  will  probably 
be  those  of  heaviest  specific  gravity — i.  e.  the 
moulds  and  yeasts. 

A  still  more  exact  method  is  that  of  Petri, 
who  uses  small  filters  of  sand  held  in  place  in  a 
wide  glass  tube  by  small  wire  nets  (Fig.  44). 
The  sand  used  is  made  to  pass  through  a 
sieve  whose  openings  are  of  known  size,  is 
heated  to  incandescence,  then  arranged  in 
the  tube  so  that  two  of  the  little  filters,  held 
in  place  by  their  wire-gauze  coverings,  are 
FIG.  44.—  superimposed.  One  or  both  ends  of  the  tube 

rfetn  I     sand  i         i       •  1  * 

filter  for  air-  are  closed  wlth  corks  having  a  narrow  glass 
examination.  tllDe-  The  apparatus  is  heated  and  sterilized 
in  a  hot-air  sterilizer,  and  is  then  ready  for 
use.  The  method  of  employment  is  very  simple.  By 
means  of  a  hand-pump  100  liters  of  air  are  made  to  pass 
through  in  from  ten  to  twenty  minutes.  The  sand  from 


BACTERIOLOGIC  EXAMINATION  OF  AIR.      167 

the  upper  filter  is  then  carefully  mixed  with  sterile 
melted  gelatin  and  poured  into  sterile  Petri  dishes,  where 
the  colonies  develop  and  can  be  counted.  Sternberg  re- 
marks that  the  chief  objection  to  the  method  is  the  pres- 
ence in  the  gelatin  of  the  slightly  opaque  sand,  which 
interferes  with  the  recognition  and  count- 
ing of  the  colonies.  This  objection  has, 
however,  been  removed  by  Sedgwick  and 
Miquel,  who  use  a  soluble  material — granu- 
lated or  pulverized  sugar — instead  of  the 
sand.  The  apparatus  used  for  the  sugar- 
experiments  differs  a  little  from  the  original 
of  Petri,  but  the  principle  is  the  same,  and 
can  be  modified  to  suit  the  experimenter. 
Petri  points  out  in  relation  to  his  method 
that  the  filter  catches  a  relatively  greater 
number  of  bacteria  in  proportion  to  moulds 
than  the  Hesse  apparatus,  which  depends 
upon  sedimentation. 

A  particularly  useful  form  of  apparatus 
is  a  granulated  sugar-filter  suggested  by 
Sedgwick  and  Tucker,  which  has  an  ex- 
pansion above  the  filter,  so  that  as  soon  as 
the  sugar  is  dissolved  in  the  melted  gela- 
tin it  can  be  rolled  out  into  a  lining  like 
that  of  an  Esmarch  tube.  This  cylindrical 
expansion  is  divided  into  squares  which 
make  the  counting  of  the  colonies  very  easy 
(Fig.  45)- 

The  number  of  germs  in  the  atmosphere 
will  naturally  be  very  variable.  Roughly,  t  , 

J  J  &      J '    tube     for     air-ex  - 

the  number  may  be  estimated  at  from  100  animation, 
to  1000  per  cubic  meter. 

In  reality,  the  bacteriologic  examination  of  air  is 
of  very  little  value,  as  so  many  possibilities  of  error 
may  occur.  Thus,  when  the  air  of  a  room  is  quiescent 
there  may  be  very  few  bacteria  in  it ;  let  some  one  walk 
across  the  floor  and  dust  at  once  rises,  and  the  number 


FIG.  45. — Sedg- 

wick's     expanded 


1 68  PATHOGENIC  BACTERIA. 

of  bacteria  is  considerably  increased :  if  the  person  be  a 
woman  with  skirts,  more  bacteria  will  probably  be  raised 
from  the  floor  than  would  be  disturbed  by  a  man  ;  if  the 
room  be  swept,  the  increase  is  enormous.  From  these 
and  similar  contingencies  it  becomes  very  difficult  to 
know  just  when  and  how  the  air  is  to  be  examined, 
and  the  value  of  the  results  is  correspondingly  lessened. 
The  most  valuable  examinations  are  those  which  aim 
at  the  discovery  of  some  definite  organism  or  organisms 
regardless  of  the  number  per  cubic  meter. 


CHAPTER  XII. 
BACTERIOLOGIC  EXAMINATION  OF  WATER. 

UNLESS  water  has  been  specially  sterilized  or  distilled 
and  received  and  kept  in  sterile  vessels,  it  always  con- 
tains some  bacteria.  The  number  will  bear  a  very  dis- 
tinct relation  to  the  amount  of  organic  matter  in  the 
water,  though  experiment  has  shown  that  certain  patho- 
genic and  non-pathogenic  bacteria  can  remain  vital  in 
perfectly  pure  distilled  water  for  a  considerable  length  of 
time.  Ultimately,  owing  to  the  lack  of  nutriment,  they 
undergo  a  granular  degeneration. 

The  majority  of  the  water-bacteria  are  bacilli,  and  as  a 


FIG.  46. — Wolf  hiigel's  apparatus  for  counting  colonies  of  bacteria  upon  plates. 


rule  they  are  non-pathogenic.  Wright,1  in  his  examina- 
tion of  the  bacteria  of  the  water  from  the  Schuylkill 
River,  found  two  species  of  micrococci,  two  species  of 
cladothrices,  and  forty-six  species  and  two  varieties  of 
bacilli.  Of  course,  at  times  the  most  virulent  forms  of 
pathogenic  bacteria — those  of  cholera  and  typhoid  fever 
— occur  in  polluted  water,  but  this  is  the  exception,  not 
the  rule. 

The  method  of  determining  quantitatively  the  number 

1  Memoirs  of  the  National  Academy  of  Sciences,  Third  Memoir. 

169 


170  PATHOGENIC  BACTERIA. 

of  bacteria  in  water  is  very  simple,  and  can  generally  be 
prosecuted  without  much  apparatus.  The  principle  de- 
pends upon  the  equal  distribution  of  a  given  quantity  of 
the  water  to  be  examined  through  a  sterile  liquid  medium, 
and  the  subsequent  solidification  of  this  medium  in  a 


FlG.  47. — Heyroth's  instrument  for  counting  colonies  of  bacteria  in  Petri  dishes. 

thin  layer,  so  that  all  the  colonies  which  develop  may 
be  counted. 

The  method,  which  originated  with  Koch,  may  be  per- 
formed with  the  Koch  plates  or  with  Petri  dishes  or 
with  Esmarch  rolls.  It  is  always  best  to  make  a  num- 
ber of  these  plate-cultures  with  different  amounts  of  the 
water  to  be  examined,  using,  for  example,  o.oi,  o.i,  0.5, 
and  i.o  c.cm.  added  to  a  tube  of  gelatin,  agar-agar,  or 
glycerin  agar-agar. 

The  exact  method  must  depend  somewhat  upon  the 
quality  of  the  water  to  be  examined.  If  the  number  of 
bacteria  per  cubic  centimeter  is  small,  large  quantities 
may  be  used,  but  if  there  are  millions  of  bacteria  in 
every  cubic  centimeter,  it  may  be  necessary  to  dilute  the 


BACTERTOLOGIC  EXAMINATION  OF   WATER.    171 

water  to  be  examined  in  the  proportion  of  i  :  10  or  i  :  100 
with  sterile  water,  mixing  well,  and  making  the  plate- 
cultures  from  the  dilutions. 

It  is  best  to  count  all  the  colonies  if  possible,  but  when 
there  are  hundreds  or  thousands  scattered  over  the  plate, 
an  average  estimation  of  a  number  of  squares  ruled  upon 
a  glass  background  (Fig.  46),  as  suggested  by  Wolf  hiigel, 
is  most  convenient.  In  his  apparatus  a  large  plate  of  glass 
is  divided  into  small  square  di- 
visions, the  diagonals  being  spe- 
cially indicated  by  color.  The 
plate  or  Petri  dish  is  stood  upon 
the  glass,  and  the  number  of 
colonies  in  a  number  of  small 
squares  is  easily  counted,  and 
the  total  number  of  colonies  es- 
timated. In  counting  the  colo- 
nies a  lens  is  indispensable. 
Special  apparatuses  have  been 
devised  for  counting  the  colo- 

Hies     in     Petri    dishes    (Fig.    47)    ,   F'^S.-Esmarch's  instrument 

T/  '    for  counting  colonies  of  bacteria 

and  in  Esmarch  tubes  (Fig.  48).   in  tubes> 

The   majority  of  the   water- 
bacteria  are  rapid  liquefiers  of  gelatin,  for  which  reason 
it   seems   better   to   employ  agar-agar   than  gelatin   for 
making  the  cultures. 

In  ordinary  hydrant-water  the  bacteria  number  from 
2-50  per  cubic  centimeter ;  in  good  pump- water,  100-500 ; 
in  filtered  water  from  rivers,  according  to  Giinther,  50-200 
are  present ;  in  unfiltered  river- water,  6000-20,000.  Ac- 
cording to  the  pollution  of  the  water  the  number  may 
reach  as  many  as  50,000,000. 

The  waters  of  wells  and  springs  are  dependent  for  their 
purity  upon  the  character  of  the  earth  or  rock  through 
which  they  filter,  and  the  waters  of  deep  wells  are  much 
more  pure  than  those  of  shallow  wells,  unless  contamina- 
tion takes  place  from  the  surface  of  the  ground. 

Ice  always  contains  bacteria  if  the  water  contained 


172  PATHOGENIC  BACTERIA. 

them  before  it  was  frozen.  In  Hudson-River  ice  Prud- 
den  found  an  average  of  398  colonies  in  a  cubic  centi- 
meter. 

A  sample  of  water  when  collected  for  examination 
should  be  placed  in  a  clean  sterile  bottle  or  in  a  her- 
metically-sealed pre-sterilized  glass  bulb,  and  must  be 
examined  as  soon  as  possible,  as  the  bacteria  multiply 
rapidly  in  water  which  is  allowed  to  stand  for  a  short 
time.  In  determining  the  species  of  bacteria  found  in 
the  water  reference  must  be  made  to  the  numerous  mono- 
graphs upon  the  subject,  and  to  tables  such  as  those 
compiled  by  Eisenberg. 

The  discovery  of  certain  important  pathogenic  bacteria, 
as  those  of  cholera  and  typhoid,  will  be  considered  under 
the  specific  headings. 

Unfortunately,  the  bacteriologic  examination  of  waters 
does  not  throw  satisfactory  light  upon  their  exact  hygi- 
enic usefulness.  Of  course,  if  cholera  or  typhoid-fever 
bacteria  are  present,  the  water  is  harmful,  but  the  quality 
of  the  water  cannot  be  gauged  by  the  number  of  bacteria 
it  contains. 

The  drinking-water  furnished  large  cities  is  not  infre- 
quently contaminated  with  sewage,  and  contains  intes- 
tinal bacteria — Bacillus  coli  communis.  For  the  ready 
determination  of  this  organism,  which  is  an  important  one 
as  an  indicator  that  the  water  is  polluted,  Smith1  has 
made  use  of  the  fermentation-tube  in  addition  to  the 
plate.  His  method  is  to  add  to  each  of  the  fermentation- 
tubes  containing  i  per  cent,  dextrose-bouillon  a  certain 
quantity  of  water.  The  evolution  of  50-60  per  cent,  of 
gas  by  the  third  day  is  a  strong  indication  that  the  colon 
bacillus  is  present.  Plates  may  be  used  to  confirm  the 
presence  of  the  bacillus,  but  are  hardly  necessary,  as 
there  is  scarcely  another  bacterium  met  with  in  water 
that  is  capable  of  producing  so  much  gas. 

Filtration  with  sand,  etc.  diminishes  the  number  of 
bacteria  for  a  time,  but,  as  the  organisms  multiply  in 

1  American  Journal  of  the  Medical  Sciences,  1895,  no,  p.  301. 


BACTERIOLOGIC  EXAMINATION  OF   WATER.    173 

the  filter,  the  benefit  is  not  permanent.  The  filters  must 
frequently  be  renewed.  Porcelain  filters  seem  to  be  the 
only  positive  safeguard,  and  even  these,  the  best  of  which 
seems  to  be  the  Pasteur-Chamberland,  allow  the  bacteria 
to  pass  through  if  used  too  long  without  renewal  or  with- 
out firing. 


CHAPTER    XIII. 
BACTERIOLOGIC  EXAMINATION  OF  SOIL. 

ALMOST  all  soil  contains  bacteria  in  its  upper  layers. 
Their  number  and  character,  however,  depend  some- 
what upon  the  surrounding  conditions.  Near  the  hab- 
itations of  men,  where  the  soil  is  cultivated,  the  ex- 
crement of  animals,  largely  made  up  of  bacteria,  is 
spread  upon  it  to  increase  its  fertility,  this  being  a  treat- 
ment which  not  only  adds  new  bacteria  to  those  already 
present,  but  also  enables  those  present  to  grow  very  much 
more  luxuriantly  because  of  the  increased 
amount  of  organic  matter  they  receive. 

The  researches  of  Fliigge,  C.  Frankel, 
and  others  show  that  the  bacteria  of  the 
soil  do  not  penetrate  very  deeply — that 
they  gradually  decrease  in  number  until 
the  depth  of  a  meter  is  reached,  then 
rapidly  diminish  until  at  a  meter  and  a 
quarter  they  rather  abruptly  cease  to  be 
found. 

Many  of  the  soil-bacteria  are  anaerobic, 
and  for  a  careful  consideration  of  them 
the  reader  must  be  referred  to  monographs 
upon  the  subject.  The  estimation  of  their 
number  seems  to  be  devoid  of  any  dis-  Fj 
tinct  practical  importance.  C.  Frankel  W'.LLentfor 
has,  however,  originated  a  very  accurate  obtaining  earth  from 
method  of  detenu  in  ing  it.  By  means  various  dePths  for 
of  a  special  boring  apparatus  (Fig.  49)  bacteriol°gic  studx- 
c-arth  can  be  secured  from  any  depth  without  digging  and 
without  danger  of  mixing  that  secured  with  that  of  the 
superficial  strata.  With  sterile  liquefied  gelatin  a  definite 

174 


BACTERIOLOGIC  EXAMINATION  OF  SOIL.     175 

amount  of  this  soil  is  mixed  thoroughly  and  the  mixture 
solidified  upon  the  walls  of  an  Esmarch  tube.  The  col- 
onies are  counted  with  the  aid  of  a  lens.  Fliigge  found 
in  virgin  earth  about  100,000  colonies  in  a  cubic  centi- 
meter. 

Samples  of  earth,  like  samples  of  water,  should  be 
examined  as  soon  as  possible  after  being  secured,  for, 
as  Giinther  points  out,  the  number  of  bacteria  changes 
because  of  the  unusual  environment,  exposure  to  increased 
amounts  of  oxygen,  etc. 

The  most  important  bacteria  of  the  soil  are  those  of 
tetanus  and  malignant  edema,  in  addition  to  which,  how- 
ever, there  are  a  great  variety  which  are  pathogenic  for 
rabbits,  guinea-pigs,  and  mice. 

In  the  "Bacteriological  Examination  of  the  Soil  of 
Philadelphia,''  &avenel l  came  to  the  conclusion  that — 

1.  Made  soils,  as  commonly  found,  are  rich  in  organic 
matter  and  excessively  damp  through  poor  drainage. 

2.  They  furnish  conditions  more  suited  to  the  multi- 
plication of  bacteria  than  do  virgin  soils,  unless  the  latter 
are  contaminated  by  sewage  or  offal. 

3.  Made  soils  contain  large  numbers  of  bacteria  per 
gram  of  many  different  species,  the  deeper  layers  being 
as  rich  in  the  number  and  variety  of  organisms  as  the 
upper  ones.     After  some  years  the  number  in  the  deeper 
layers  probably  becomes  proportionally  less.     Made  soils 
are  more  likely  than  others  to  contain  pathogenic  bacteria. 

In  71  cultures  that  were  isolated  and  carefully  studied 
by  Ravenel,  there  were  two  cocci,  one  sarcina,  and  five 
cladothrices;  all  the  others  were  bacilli. 

1  Memoirs  of  the  National  Academy  of  Sciences,  First  Memoir,  1896. 


CHAPTER    XIV. 
TO  DETERMINE  THE  THERMAL  DEATH-POINT. 

SEVERAL  methods  may  be  employed  for  this  purpose. 
Roughly,  it  may  be  done  by  keeping  a  bouillon-culture  of 
the  micro-organism  to  be  studied  in  a  water-bath  whose 
temperature  is  gradually  increased  from  that  of  the  body 

to  75°  C. 

Into  a  fresh  bouillon-culture  thus  exposed  to  heat,  the 
experimenter  cautiously,  and  at  given  intervals,  intro- 
duces a  platinum  loop  or  a  capillary  pipette,  and  with- 
draws a  drop  of  the  culture  which  he  inoculates  into 
fresh  bouillon  and  stands  aside  to  grow.  It  is  economy 
to  make  the  transplantations  rather  infrequently  at  first 
and  frequently  later  on  in  the  experiment,  when  the  tem- 
perature is  ascending.  In  an  ordinary  determination  it 
would  be  well  to  make  a  transfer  at  40°  C.,  one  at  45°  C., 
another  at  50°,  still  another  at  55°,  and  then  beginning  at 
60°  make  one  for  every  additional  degree  up  to  75°  C. 
or  above.  The  day  following  the  experiment  it  will  be 
observed  that  all  the  cultures  grow  except  those  heated 
beyond  a  certain  point,  as  60°  C.  and  upward,  when  it 
can  properly  be  concluded  that  60°  C.  is  the  thermal 
death-point.  If  all  the  transplantations  grow,  of  course 
the  maximum  temperature  that  the  bacteria  can  endure 
was  not  reached,  and  the  experiment  must  be  performed 
again  with  higher  temperatures. 

When  more  accurate  information  is  desired,  and  one 
wishes  to  know  how  long  the  micro-organism  can  endure 
some  such  temperature  as  60°  C.  without  losing  its  vital- 
ity, a  dozen  or  more  bouillon-tubes  may  be  inoculated 
with  the  germ  to  be  studied,  and  stood  in  the  water-bath 
at  the  temperature  to  be  investigated.  The  first  can  be 

176 


TO  DETERMINE    THERMAL  DEATH-POINT.    177 

removed  as  soon  as  it  is  certainly  heated  through,  another 
in  five  minutes,  another  in  ten  minutes,  or  at  whatever 
intervals  the  thought  and  experience  of  the  experimenter 
shall  suggest. 

In  both  of  the  described  procedures  one  must  be  care- 
ful that  the  temperature  in  the  test-tube  is  identical  with 
that  of  the  water  in  the  bath.  There  is  no  reason  why  a 
sterile  thermometer  should  not  be  placed  in  the  heated 
tube  in  the  first  case,  and  in  the  second  experiment  one 
of  the  test-tubes  exposed  under  conditions  similar  to  the 
others  might  contain  a  thermometer  which  would  show 
the  temperature  of  the  contents  of  the  tube  containing  it, 
and  so  be  an  index  of  the  rest. 

Another  method  of  accomplishing  the  same  end  is  to 
use  Sternberg's  bulbs.  These  are  small  glass  bulbs 
blown  on  one  end  of  a  piece  of  glass  tubing,  which  is 
subsequently  drawn  out  to  capillarity  at  the  opposite  end. 
If  such  a  bulb  be  heated,  and  its  capillary  tube  dipped 
into  inoculated  bouillon,  in  cooling,  the  fluid  is  drawn  in 
so  as  to  fill  it  one-third  or  one-half.  A  number  of  these 
tubes  are  filled  in  this  manner  with  freshly  inoculated 
culture-medium  and  then  floated,  tube  upward,  upon 
a  water-bath  whose  temperature  is  gradually  elevated, 
the  bulbs  being  removed  from  time  to  time  as  the 
required  temperatures  are  reached.  Of  course,  as  the 
bulbs  are  already  inoculated,  all  that  is  necessary  is  to 
stand  them  aside  for  a  day  or  two,  and  observe  whether 
or  not  the  bacteria  grow,  iudging  the  death-point  exactly 
as  in  the  other  case. 

To  Determine  the  Antiseptic  and  Germicidal  Value 
of  Reagents. — There  are  various  methods  whose  modi- 
fications can  be  elaborated  according  to  the  extent  and 
thoroughness  of  the  investigation  to  be  made. 

I.  The  Antiseptic  Value. — Remembering  that  an  anti- 
septic is  a  substance  that  inhibits  bacterial  growth,  the 
method  that  will  at  once  suggest  itself  is  that  of  adding 
varying  quantities  of  the  antiseptic  to  be  investigated  to 
culture-media  in  which  the  bacteria  are  subsequently 
12 


1 78  PA  THOGENIC  BA  CTERIA. 

planted.  It  is  always  well  to  use  a  considerable  number 
of  tubes.  Bouillon  is  generally  employed.  If  the  anti- 
septic is  non-volatile,  it  may  be  added  before  sterilization, 
which  is  to  be  preferred;  but  if  it  is  volatile,  it  must  be 
added  by  means  of  a  sterile  pipette,  with  the  greatest 
precaution  as  regards  asepsis,  immediately  before  the  test 
is  to  be  made.  Control-experiments — i.  e.  without  the 
addition  of  the  antiseptic — should  always  be  made. 

The  results  of  antiseptic  action  are  two:  retardation  of 
growth  and  complete  inhibition  of  growth.  As  the  tubes 
used  for  the  study  of  the  antiseptic  are  watched  in  their 
development,  it  will  usually  be  noticed  that  those  con- 
taining very  small  quantities  develop  almost  as  rapidly  as 
the  control-tubes;  those  containing  more,  a  little  more 
slowly;  those  containing  still  more,  very  slowly,  until  at 
last  there  comes  at  time  when  the  growth  is  not  deferred, 
but  prevented. 

Sternberg  points  out  that  certain  circumstances  may 
modify  the  results  obtained.  They  are: 

1.  The  composition  of  the  nutrient  media,  with  which 
the  antiseptic  may  be  incompatible. 

2.  The  nature  of  the  test-organism,  no  two  organisms 
being  exactly  alike  in  their  susceptibility. 

3.  The  temperature  at  which  the  experiment  is  con- 
ducted,  a  relatively  greater  amount   of  the   antiseptic 
being  necessary  at  temperatures  favorable  to  the  organ- 
ism than  at  temperatures  unfavorable. 

4.  The  presence   of  spores   which   are   always  more 
resistant  than  the  asporogenous  forms. 

II.  The  Germicidal  Value. — Koch's  original  method  of 
doing  this  was  to  dry  the  micro-organisms  upon  sterile 
shreds  of  linen  or  silk,  and  then  soak  them  for  varying 
lengths  of  time  in  the  germicidal  solution.  After  the 
bath  in  the  reagent  the  threads  were  washed  in  clean, 
sterile  water  and  then  transferred  to  fresh  culture- 
media,  and  their  growth  or  failure  to  grow  observed.  It 
will  be  observed  that  this  method  is  aimed  at  the  deter- 
mination of  the  time  in  which  a  certain  solution  will  kill. 


TO  DETERMINE    THERMAL  DEATH-POINT.    179 

Sternberg  suggested  a  method  in  which  the  time  should 
remain  constant  (two  hours'  exposure),  and  the  object  be 
the  determination  of  the  exact  dilution  of  the  reagent 
required  to  destroy  the  bacteria.  "Instead  of  subjecting 
a  few  of  the  test-organisms  attached  to  a  silk  thread  to 
the  action  of  the  disinfecting  agent,  a  certain  quantity  of 
the  recent  culture — usually  5  c.cm. — has  been  mixed 
with  an  equal  quantity  of  a  standard  solution  of  the 
germicidal  agent,  .  .  .  and  after  two  hours'  contact  one 
or  two  ose-fuls  would  be  introduced  into  a  suitable  nutri- 
ent medium  to  test  the  question  of  disinfection." 

A  very  simple  and  popular  method  of  determining  the 
germicidal  value  is  to  make  a  series  of  dilutions  of  the 
reagent  to  be  tested;  add  to  each  a  couple  of  loopfuls  of 
a  fresh  liquid  culture,  and  at  varying  intervals  of  time 
transfer  a  loopful  to  fresh  culture-media.  By  a  little 
ingenuity  this  method  may  be  made  to  yield  information 
as  to  both  time  and  strength. 

When  it  is  desired  to  secure  information  concerning 
the  progress  of  the  germicidal  action  of  reagents,  body- 
fluids,  etc.,  especially  in  the  unusual  and  interesting 
cases  in  which  the  material  subjected  to  the  test  may 
exert  a  restraining  action  for  a  time  only,  or  bring  about 
destruction  of  some  or  many,  but  not  all  of  the  germs, 
the  use  of  the  Petri  dish  can  be  introduced. 

For  example,  it  is  desired  to  determine  whether  a 
blood-serum  is  germicidal  or  not.  Into  about  5  c.cm.  of 
the  serum  contained  in  a  test-tube,  two  or  three  loopfuls 
of  any  desired  bacterium,  in  liquid  culture,  are  added. 
The  tube  is  well  agitated  and  immediately  one  loopful  is 
transferred  to  a  tube  of  melted  gelatin,  distributed 
through  it,  and  poured  into  a  Petri  dish.  After  one 
minute  the  operation  is  repeated,  in  five  minutes  again, 
and  so  on  as  often  as  is  desired. 

The  dishes  are  stood  away  until  the  bacteria  develop 
into  colonies,  which  are  then  counted  with  a  Wolfhiigel 
apparatus.  On  the  first  dish  there  may  be  100  colonies; 
on  the  second,  80;  on  the  third,  50;  on  the  fourth,  20;  on 


l8o  PATHOGENIC  BACTERIA. 

the  fifth,  30;  on  the  sixth,  150;  on  the  seventh,  1000, 
etc. ;  indicating  that  the  serum  exerted  a  destructive 
action  upon  some  but  not  all  of  the  bacteria,  and  that 
this  power  disappeared  after  the  lapse  of  a  certain  time, 
allowing  the  bacteria  to  develop  ad  libitum. 

When  the  germicide  to  be  studied  is  a  gas,  as  in  the 
case  of  sulphurous  acid  or  formaldehyd,  a  different 
method  must,  of  course,  be  adopted. 

It  may  be  sufficient  simply  to  place  a  few  test-tube  cul- 
tures of  various  bacteria,  some  with  plugs  in,  some  with 
plugs  out,  in  a  closed  room  in  which  the  gas  is  afterward 
evolved.  The  germicidal  action  is  shown  by  the  failure 
of  the  cultures  to  grow  upon  transplantation  to  fresh  cul- 
ture-media. This  crude  method  may  be  supplemented 
by  an  examination  of  the  dust  of  the  room.  Pledgets 
of  sterile  cotton  are  rubbed  upon  the  floor,  washboard, 
or  any  dust-collecting  surface  present,  and  subsequently 
dropped  into  culture-media.  Failure  of  growth  under 
such  circumstances  is  very  certain  evidence  of  good  dis- 
infection. These  tests  are,  however,  very  severe,  for  in 
the  cultures  there  are  immense  numbers  of  bacteria  in 
the  deeper  portions  of  the  bacterial  mass  upon  which  the 
gas  has  no  opportunity  to  act,  and  in  the  dust  there  are 
many  sporogenous  organisms  of  extreme  resisting  power. 
Failure  to  kill  all  the  germs  exposed  in  such  manner  is 
no  indication  that  the  vapor  cannot  destroy  all  the  ordi- 
nary pathogenic  organisms. 

More  refined  is  the  method  of  saturating  sterile  sand 
or  fragments  of  blotting-paper  or  absorbent  cotton  with 
cultures  and  exposing  them,  moist  or  dry,  to  the  action 
of  the  gas.  Such  materials  are  best  made  ready  in  Petri 
dishes,  which  are  opened  immediately  before  and  closed 
i  in  mediately  after  the  experiment.  A  piece  of  cotton  or 
blotting-paper  or  a  little  sand  transferred  to  fresh  culture- 
media  will  not  give  any  growth  where  the  disinfection  has 
been  thorough.  By  transplanting  from  different  depths, 
the  sand  may  be  used  incidently  to  show  to  what  depth 
the  gas  is  capable  of  penetrating. 


TO  DETERMINE    THERMAL  DEATH-POINT.    181 

Easier  of  execution,  but  rather  more  severe,  is  a 
method  in  which  cover-glasses  are  employed.  A  num- 
ber of  them  are  spread  with  cultures  of  various  bacteria, 
allowed  to  dry,  and  then  exposed  to  the  gas  as  long  as 
required.  The  cover-glasses  are  afterward  dropped  into 
culture-media  to  permit  the  growth  of  the  germs  not 
destroyed. 

Animal-experiments  may  also  be  employed  to  deter- 
mine whether  or  not  a  germ  that  has  survived  exposure  to 
the  action  of  reagents  has  its  pathogenic  power  destroyed. 
An  excellent  example  of  this  is  seen  in  the  case  of  the 
anthrax  bacillus,  a  virulent  form  of  which  will  kill  rab- 
bits, but  after  being  grown  in  media  containing  an 
insufficient  amount  of  a  germicide  to  kill  it  will  often 
lose  its  rabbit-killing  power,  though  still  able  to  fatally 
infect  guinea-pigs,  or  may  lose  its  virulence  for  both 
rabbits  and  guinea-pigs,  though  still  able  to  kill  white 
mice. 


PART  II.     SPECIFIC  DISEASES  AND  THEIR 
BACTERIA. 


A.     THE   PHLOGISTIC   DISEASES. 


I.     THE   ACUTE   INFLAMMATORY  DISEASES. 


CHAPTER    I. 
SUPPURATION. 

SUPPURATION  was  at  one  time  supposed  to  be  an 
inevitable  outcome  of  the  majority  of  wounds,  and, 
although  bacteria  were  observed  in  the  discharges,  the 
old  habit  of  thought  and  insufficiency  of  information 
caused  most  surgeons  to  believe  that  they  were  sponta- 
neously developed  there. 

Lord  Lister,  whose  name  we  cannot  sufficiently  honor, 
conceived  that  Pasteur's  observations  upon  the  germs  of 
life  floating  in  the  atmosphere,  if  they  explained  the  con- 
tamination of  his  sterile  infusions,  might  also  explain 
the  changes  in  wounds,  and  upon  this  idea  based  that 
most  successful  system  of  treatment  known  as  "  antisep- 
tic surgery." 

The  further  development  of  antiseptic  surgery,  and  the 
extremes  to  which  it  was  carried  by  specialists,  almost 
attain  to  the  ridiculous,  for  not  only  were  the  hands  of 
the  operator,  his  instruments,  sponges,  sutures,  ligatures, 
and  dressings  kept  constantly  saturated  with  irritating 
germicidal  solutions,  but  at  one  time  the  air  over  the 
wound  was  carefully  saturated  with  pulverized  antiseptic 
lotions  during  the  whole  operation  by  means  of  a  steam 
atomizer.  This  rather  monstrous  outcome  of  the  appli- 
cation of  Lister's  system  to  surgery  was  the  very  natural 
result  of  the  erroneous  idea  that  the  germs  which  cause 

182 


SUPPURA  TION.  183 

the  suppurative  changes  in  wounds  entered  the  exposed 
tissues  principally  from  the  atmosphere,  and  that  the 
hands  and  instruments  of  the  operator,  while  certainly 
means  of  infection,  were  secondary  in  importance  to  it. 

The  researches  of  more  recent  date,  however,  have 
shown  not  only  that  the  atmosphere  cannot  be  disin- 
fected, but  also  that  the  air  of  ordinarily  quiet  rooms, 
while  containing  the  spores  of  numerous  saprophytic 
organisms,  very  rarely  contains  many  pathogenic  bac- 
teria. We  now  also  know  that  a  direct  stream  of  air, 
such  as  is  generated  by  an  atomizer,  causes  more  bacteria 
to  be  conveyed  into  a  wound  than  would  ordinarily  fall 
upon  it,  thereby  increasing  instead  of  lessening  the  dan- 
ger of  infection.  It  may  therefore  be  stated,  with  a 
reasonable  amount  of  certainty,  that  the  atmosphere  is 
rarely  an  important  factor  in  the  process  of  suppuration. 

We  have  already  called  attention  to  the  fact  that 
various  micro-organisms  are  so  intimate  in  their  relation 
to  the  skin  that  it  is  almost  impossible  to  get  rid  of  them, 
and  have  cited  in  this  relation  the  experiments  of  Welch, 
Robb,  and  Ghriskey,  whose  method  of  disinfecting  the 
hands  has  been  recommended  as  the  best.  The  investi- 
gations of  these  observers  have  shown  that,  no  matter 
how  rigid  the  disinfection  of  the  patient's  skin,  the 
cleansing  of  the  operator's  hands,  the  sterilization  of 
the  instruments,  and  the  precautions  exercised,  a  certain 
number  of  wounds  in  which  sutures  are  employed  will 
always  suppurate.  The  cause  of  the  suppuration  is  a 
matter  of  vast  importance  in  surgery  and  in  surgical  bac- 
teriology, yet  it  is  one  which  it  is  impossible  to  remove. 
We  carry  it  constantly  with  us  upon  our  skins. 

STAPHYLOCOCCUS  EPIDERMIDIS  ALBUS. 
Welch  has  described,  under  the  name  Staphylococcus 
epidermidis  albus,  a  micrococcus  which  seems  to  be  habit- 
ually present  upon  the  skin,  not  only  upon  the  surface, 
but  also  deep  down  in  the  Malpighian  layer.  He  is  of 
the  opinion  that  it  is  the  same  organism  which  is  familiar 


184  PATHOGENIC  BACTERIA. 

to  us  under  the  name  of  Staphylococcus  pyogenes  albus, 
but  in  an  attenuated  condition.  If  his  opinion  be  correct, 
and  we  have  seated  deeply  in  our  derm  a  coccus  which 
can  at  times  cause  abscess-formation,  the  conclusions  of 
Robb  and  Ghriskey,  that  sutures  of  catgut  when  tightly 
drawn  may  be  a  cause  of  skin-abscesses  by  predisposing 
to  the  development  of  this  organism,  are  certainly  justi- 
fiable. 

Not  only  does  the  coccus  occur  in  the  attenuated  form 
described,  but  we  have  very  commonly  present  upon  the 
skin,  generally  as  a  harmless  saprophyte,  the  important 
Staphylococcus  pyogenes  albus,  which  is  a  common  cause 
of  suppuration. 

STAPHYLOCOCCUS  PYOGENES  ALBUS. 

Although,  as  stated,  the  Staphylococcus  pyogenes  albus 
is  a  common  cause  of  suppuration,  it  rarely  occurs  alone, 
the  studies  of  Passet  showing  that  in  but  4  out  of  33  cases 
which  he  investigated  was  this  coccus  found  by  itself. 
When  pure  cultures  of  the  coccus  are  injected  subcu- 
taneously  into  rabbits  and  guinea-pigs,  abscesses  some- 
times result ;  sometimes  there  is  no  result.  Injected 
into  the  circulation  of  these  animals,  the  staphylococci 
sometimes  cause  septicemia,  and  after  death  can  be  found 
in  the  capillaries,  especially  of  the  kidneys.  From  these 
illustrations  it  will  be  seen  that  the  organism  is  feebly 
pathogenic. 

In  its  vegetative  characteristics  the  Staphylococcus 
albus  is  almost  identical  with  the  species  next  to  be  de- 
scribed, but  differs  from  it  in  that  there  is  no  golden  color 
produced.  Upon  the  culture-media  it  grows  white. 

STAPHYLOCOCCUS  PYOGENES  AUREUS. 
Generally  present  upon  the  skin,  though  in  smaller 
numbers,  is  the  dangerous  and  highly  virulent  Staphyh- 
comts py,^t  >i<  s  tinn-ns  (Fig.  50),  or  "golden  Staphylococ- 
cus "  of  Rosenbach.  As  the  morphology  of  this  organ- 
ism, and  indeed  the  generality  of  its  characters,  are 


SUPPURA  TION.  1 85 

identical  with  those  of  the  preceding  species,  it  seems 
convenient  to  describe  them  together,  pointing  out  such 


FIG.  50. — Staphylococcus  pyogenes  aureus,  from  an  agar-agar  culture;    x  1000 

(Gunther). 

differences  as  occur  step  by  step.  In  doing  this,  how- 
ever, it  must  not  be  forgotten  that,  although  the  Staphy- 
lococcus albus  has  been  described  first,  the  Staphylococcus 
aureus  is  the  more  common  organism  of  the  suppurative 
diseases. 

Although  they  had  been  seen  earlier  by  several  ob- 
servers, the  staphylococci  were  not  isolated  and  care- 
fully described  until  1884,  when  Rosenbach  worked  upon 
them.  The  results  of  his  study,  followed  by  Passet  and 
a  host  of  others,  have  now  given  us  pretty  accurate 
information  about  them. 

The  cocci  are  distributed  rather  sparingly  in  nature, 
seeming  not  to  find  a  purely  saprophytic  existence  a 
suitable  one.  They  occur,  however,  wherever  man  and 
animals  have  been,  and  can  be  found  in  the  dust  of 
houses,  hospitals,  and  especially  surgical  wards  where 
proper  precautions  are  not  exercised.  They  are  common 
upon  the  skin,  they  live  in  the  nose,  mouth,  eyes,  and 
ears  of  man,  they  are  nearly  always  beneath  the  finger- 
nails, and  they  sometimes  occur  in  the  feces,  especially 
in  children. 


186  PATHOGENIC  BACTERIA. 

The  cocci  are  rather  small,  measuring  about  0.7  //  in 
diameter.  When  examined  in  a  delicately-stained  con- 
dition the  organisms  may  be  seen  to  consist  of  hemi- 
spheres separated  from  each  other  by  a  narrow  interval. 
The  contiguous  surfaces  are  flat,  thus  differing  from 
the  gonococcus,  whose  contiguous  surfaces  are  concave. 
The  grouping  is  not  very  characteristic.  In  both  liquid 
and  solid  culture-media  the  organisms  either  occur  in 
solid  masses  or  are  evenly  distributed.  It  is  only  in  the 
organs  or  tissues  of  a  diseased  animal  that  it  is  possible  to 
say  that  a  true  staphylococcus  grouping  is  present. 

The  organism  stains  brilliantly  with  aqueous  solu- 
tions of  the  anilin  dyes.  In  tissues  it  can  be  beautifully 
stained  by  Gram's  method. 

The  staphylococci  grow  well  either  in  the  presence  or 
absence  of  oxygen  at  a  temperature  above  18°  C.,  the 
most  rapid  development  being  at  about  37°  C.  Upon  the 
surface  of  gelatin  plates  small  whitish  points  can  be 
observed  in  forty-eight  hours  (Fig.  51).  These  rapidly 


FIG.  51.— Staphylococcus  pyogenes  aureus:  colony  two  days  old,  seen  upon  an 
agar-agar  plate ;    x  40  ( Heim). 

extend  to  the  surface  and  cause  extensive  liquefaction. 
Hand  in  hand  with  the  liquefaction  is  the  formation  of 
an  orange  color,  which  is  best  observed  at  the  centre  of 
the  colony.  Under  the  microscope  the  colonies  appear 


SUPPURA  TION. 


187 


as  round  disks  with  circumscribed,  smooth  edges.  They 
are  distinctly  granular  and  dark-brown.  When  the  col- 
onies are  grown  upon  agar-agar  plates  the  formation  of 
the  pigment  is  much  more  distinct. 

In  gelatin  punctures  the  growth  occurs  along  the  whole 
length  of  the  needle-track,  and  causes  an  extensive  lique- 
faction in  the  form  of  a  long,  narrow,  blunt-pointed, 
inverted  cone  (Fig.  52)  full  of  clouded  liquid,  at  the  apex 


FIG.  52. — Staphylococcus  pyogenes  aureus :    puncture-culture  three   days  old 
in  gelatin   (Frankel  and  Pfeiffer). 

of  which  a  collection  of  golden  or  orange-yellow  precipi- 
tate is  always  present.  It  is  this  precipitate  in  particu- 
lar that  gives  the  organism  its  name,  "golden  staphylo- 
coccus." 

The  most  characteristic  growth  is  upon  agar-agar. 
Along  the  whole  line  of  inoculation  an  orange-yellow, 
moist,  shining  growth  occurs.  When  the  growth  takes 
place  rapidly,  as  in  the  incubator,  it  exceeds  the  rapidity 


1 88  PA  THOGENIC  BA  CTERIA . 

of  color-production,  so  that  the  centre  of  the  growth  is 
distinctly  golden  ;  the  edges  may  be  white. 

Upon  potato  the  growth  is  luxuriant,  producing  an 
orange-yellow  coating  over  a  large  part  of  the  surface. 
The  potato-cultures  give  off  a  sour  odor. 

When  grown  in  bouillon  the  organism  causes  a  diffuse 
cloudiness. 

In  milk  coagulation  takes  place,  and  is  followed  by 
gradual  digestion  of  the  casein. 

The  Staphylococcus  albus  is  exactly  the  same  as  the 
aureus,  with  the  exception  that  in  all  media  it  is  con- 
stantly colorless. 

Experiments  have  shown  that  the  Staphylococcus 
aureus,  like  its  congener,  the  albus,  exists  in  an  atten- 
uated form,  and  there  is  every  reason  to  believe  that  in 
the  majority  of  instances  it  inhabits  the  surface  of  the 
body  in  that  condition. 

When  virulent  the  golden  Staphylococcus  is  a  danger- 
ous and  often  deadly  organism.  Its  pathogeny  among 
animals  is  decided.  When  introduced  subcutaneously, 
abscesses  almost  invariably  follow,  except  in  a  certain 
few  comparatively  immune  species,  and  not  infrequently 
lead  to  a  fatal  termination.  In  such  cases  the  organisms 
may  be  cultivated  from  the  blood  of  the  large  vessels, 
though  by  far  the  greater  number  collect  in,  and  fre- 
quently obstruct,  the  capillaries.  In  the  lungs  and 
spleen,  and  still  more  frequently  in  the  kidneys,  infarcts 
are  formed  by  the  bacterial  emboli.  The  Malpighian 
tufts  of  the  kidneys  sometimes  are  full  of  cocci,  and 
become  the  centres  of  small  abscesses. 

The  coccus  is  almost  equally  pathogenic  for  man, 
though  the  fatal  outcome  is  much  more  rare.  It  enters 
the  system  through  scratches,  punctures,  or  abrasions, 
and  when  virulent  generally  causes  an  abscess,  as  various 
experimenters  who  inoculated  themselves  have  discov- 
ered to  their  cost.  Garre  applied  the  organism  in  pure 
culture  to  the  uninjured  skin  of  his  arm,  and  in  four 
days  developed  a  large  carbuncle  with  a  surrounding 


SUPPURA  TION.  1 89 

zone  of  furuncles.  Bockhart  suspended  a  small  portion 
of  an  agar-agar  culture  in  salt-solution,  and  scratched  it 
gently  into  the  deeper  layers  of  the  skin  with  his  finger- 
nail ;  a  furuncle  developed.  Bumm  injected  the  coccus 
suspended  in  salt-solution  beneath  his  skin  and  that  of 
several  other  persons,  and  produced  an  abscess  in  every 
case. 

The  Staphylococcus  aureus  is  not  only  found  in  the 
great  majority  of  furuncles,  carbuncles,  abscesses,  and 
other  inflammatory  diseases  of  the  surface  of  the  body, 
but  also  plays  an  important  role  in  a  number  of  deeply- 
seated  diseases  of  the  internal  organs.  Becker  and  others 
obtained  it  from  the  pus  of  osteomyelitis,  demonstrating 
that  if,  after  fracturing  or  crushing  a  bone,  the  staphylo- 
coccus  was  injected  into  the  circulation,  osteomyelitis 
would  result.-  Numerous  bacteriologists  have  demon- 
strated its  presence  in  ulcerative  endocarditis.  Rodet 
has  been  able  to  produce  osteomyelitis  without  previ- 
ous injury  to  the  bones ;  Rosenbach  was  able  to  produce 
ulcerative  endocarditis  by  injecting  some  of  the  staphy- 
lococci  into  the  circulation  in  animals  whose  cardiac 
valves  had  been  injured  by  a  sound  passed  into  the 
carotid  artery  ;  and  Ribbert  has  shown  that  the  injection 
of  cultures  of  the  organism  may  cause  the  valvular  lesion 
without  the  preceding  injury. 

The  Staphylococcus  aureus  is  an  easy  organism  to  ob- 
tain, and  can  be  secured  by  plating  out  a  drop  of  pus  in 
gelatin  or  in  agar-agar.  Such  a  preparation,  however, 
generally  does  not  contain  the  Staphylococcus  aureus 
alone,  but.  shows  colonies  of  the  Staphylococcus  albus  as 
well.  In  addition  to  these  two  principal  forms,  one 
sometimes  discovers  an  organism  identical  with  the  pre- 
ceding, except  that  its  growth  on  agar-agar  and  potato 
is  of  a  brilliant  lemon-yellow  color,  and  its  pathogeny  for 
animals  much  less.  This  is  the  Staphylococctis  citreus  of 
Passet.  It  is  not  quite  so  common,  and  not  so  patho- 
genic as  the  others,  and  consequently  much  less  im- 
portant. 


190 


PATHOGENIC  BACTERIA. 


STREPTOCOCCUS  PYOGENES. 

Another  organism  whose  colonies  are  frequently  ob- 
tained from  the  pus  containing  the  staphylococci  is  the 
Streptococcus  pyogenes  of  Rosenbach  (Fig.  53).  It  was 
found  by  him  in  18  of  33  cases  of 
suppurative  lesions  studied,  fifteen 
times  alone  and  five  times  with  the 
Staphylococcus  aureus.  It  is  a 
spherical  organism  of  variable  size 
(0.4-1  //  in  diameter),  constantly 


FIG.  53. — Streptococcus  pyogenes,  from  the  pus 
taken  .from  an  abscess;  x  1000  (Frankel  and 
Pfeiffer). 


FIG.  54. — Streptococ- 
cus pyogenes :  culture 
upon  agar-agar  two  days 
old  (Frankel  and  Pfeif- 
fer). 


associated  in  pairs  and  chains  of  from  four  to  twenty  in- 
dividuals. A  special  variety  of  it,  known  as  Streptococ- 
cus longus,  sometimes  forms  chains  of  more  than  one 
hundred  members. 

The  organism  stains  well  with  ordinary  aqueous  solu- 
tions of  the  anilin  dyes,  and  also  by  Gram's  method.  Like 
the  coccus  already  described,  it  is  not  motile  and  does  not 
seem  to  form  spores,  though  sometimes  a  large  individual 
— much  larger  than  the  others  in  its  chain — may  be  ob- 
served, and  may  suggest  the  thought  of  arthro-sporulation. 


SUPPURA  TION.  191 

Upon  gelatin  plates  very  small  colonies  of  translucent 
appearance  are  observed.  When  superficial,  they  spread 
out  to  form  flat  disks  about  0.5  mm.  in  diameter.  The 
microscope  shows  them  to  be  irregular  and  granular,  to 
have  a  slightly  yellowish  color,  and  to  have  numerous 
irregularities  around  the  edges,  due  to  projecting  chains 
of  the  cocci.  No  liquefaction  occurs. 

In  gelatin  puncture-cultures  no  liquefaction  is  observed. 
The  minute  spherical  colonies  grow  along  the  whole 
needle-track  and  form  a  slightly  opaque  granular  line. 

Upon  agar-agar  an  exceedingly  delicate  transparent 
growth  develops  slowly  along  the  line  of  inoculation. 
It  consists  of  almost  transparent,  colorless  small  colonies 
which  do  not  become  confluent. 

The  growth  upon  blood-serum  much  resembles  that 
upon  agar-agar.  The  streptococcus  does  not  seem  to 
grow  upon  potato. 

In  bouillon  the  cocci  develop  rather  slowly,  seeming 
to  prefer  a  neutral  or  feebly  acid  reaction.  The  culture- 
medium  remains  clear,  while  numerous  small  flocculi  are 
suspended  in  it.  When  the  flocculi-formation  is  very 
distinct  the  name  Streptococcus  conglomerates  is  used 
to  describe  the  organism.  These  masses  sometimes  ad- 
here to  the  sides  of  the  tube;  sometimes  they  form  a  sedi- 
ment. Rarely,  there  is  general  clouding  of  the  medium 
(Streptococcus  diffusus). 

In  mixtures  of  bouillon  and  blood-serum  or  ascitic 
fluid  the  streptococcus  grows  much  better,  especially  at 
incubation  temperatures,  and  in  such  mixtures  the  lux- 
uriant development  causes  the  liquid  to  appear  clouded. 

The  organism  seems  to  grow  well  in  milk,  which  is 
coagulated  and  digested. 

The  streptococcus  is  not  very  sensitive  to  acids,  and 
can  be  grown  quite  well  in  media  with  a  slightly  acid 
reaction. 

Sternberg  found  that  the  streptococci  succumb  to  a 
temperature  of  52°-54°  C.  continued  for  ten  minutes. 

Their   vitality  in   culture   is   not  great.     Unless  fre- 


PATHOGENIC  BACTERIA. 

quently  transplanted  they  die.  In  bouillon  they  are  said 
to  die  in  five  to  ten  days.  On  solid  media  they  seem 
to  retain  their  vegetative  and  pathogenic  powers  much 
longer.  They  resist  drying  well.  Their  growth  in  arti- 
ficial media  is  accompanied  by  the  production  of  an 
acid  which  probably  acts  destructively  upon  the  bacteria 
themselves. 

The  Streptococcus  pyogenes  is  generally  not  very  patho- 
genic for  animals.  Subcutaneous  injections  into  mice 
and  rabbits  are,  as  a  rule,  without  either  general  or  local 
manifestations  of  importance.  If,  however,  an  ear  of  a 
rabbit  is  carefully  inoculated  with  a  small  amount  of  a 
pure  culture,  a  small  patch  resembling  erysipelas  usually 
results.  The  disturbance  passes  away  in  a  few  days  and 
the  animal  recovers. 

If,  however,  the  streptococcus  is  highly  virulent,  the 
rabbit  dies  in  from  twenty-four  hours  to  six  days  from 
a  general  septicemia.  The  cocci  may  be  found  in  large 
numbers  in  the  heart's  blood  and  in  the  organs.  In  less 
virulent  cases  minute  disseminated  abscesses  are  some- 
times found. 

According  to  Marmorek,1  the  virulence  can  be  increased 
to  a  remarkable  degree  by  rapid  passage  through  rabbits, 
and  maintained  by  the  use  of  a  culture-medium  consist- 
ing of  three  parts  of  human  blood-serum  and  one  of 
bouillon.  The  blood  of  the  ass,  and  ascitic  and  chest 
fluids  may  also  be  used.  By  these  means  Marmorek  suc- 
ceeded in  intensifying  the  virulence  of  his  culture  to  such 
a  degree  that  one  hundred  millionth  of  a  c.cm.  injected 
into  the  ear  vein  was  fatal  to  a  rabbit. 

Petruschky 2  found  the  virulence  of  the  culture  to  be 
well  retained  if  the  culture  was  planted  in  gelatin,  trans- 
planted every  five  days,  and  when  grown  kept  on  ice. 

Hoist3  succeeded  in  keeping  an  exceedingly  virulent 
Streptococcus  brevis  on  artificial  culture-media  for  eight 

1  Ann.  dt  /'/«.?/.  /W<v/r.  Tome  ix.,  No.  7,  July  25,  1895,  p.  593. 

2  Ctntralbl.  fiir  Bakt.  und  Parasitenk.,  Bd.  xviii.,  No.  16,  May  4,  1895,  P- 
551-  s  Ibid.,  Bd.  xix.,  No,  ii,  Mar.  21,  1896. 


SUPPURA  TION.  193 

years  without  any  particular  precautions  and  found  its 
virulence  unchanged. 

Probably  the  virulence  and  attenuation  are  peculiarities 
of  the  organism  itself. 

Dried  streptococci  are  said  by  Frosch  and  Kolle  to  re- 
tain their  energies  longer  than  those  growing  on  culture- 
media.1 

Like  the  staphylococci,  the  streptococcus  is  frequently 
associated  with  internal  diseases,  and  has  been  found  in 
erysipelas,  ulcerative  endocarditis,  periostitis,  otitis,  men- 
ingitis, emphysema,  pneumonia,  lymphangitis,  phleg- 
mons, sepsis,  and  in  the  uterus  in  cases  of  infective  puer- 
peral endometritis.  In  man  the  streptococci  occur  in  the 
most  active  forms  of  suppuration.  Its  relation  to  diph- 
theria is  of  interest,  for,  while,  in  all  probability,  the 
great  majority  of  cases  of  pseudomembranous  angina  are 
caused  by  the  Klebs-Loffler  bacillus,  yet  an  undoubted 
number  of  cases  are  met  with  in  which,  as  in  Prudden's 
24  cases,  no  diphtheria  bacilli  can  be  found,  but  which 
seem  to  be  caused  by  a  streptococcus  exactly  resembling 
that  under  consideration. 

There  is  no  clinical  difference  in  the  picture  of  the 
throat-lesion  produced  by  the  two  organisms,  and  the 
only  positive  method  of  diagnosticating  the  one  from 
the  other  is  by  means  of  a  careful  bacteriologic  examina- 
tion. Such  an  examination  should  always  be  made,  as  it 
has  much  weight  in  connection  with  the  treatment.  Of 
course,  in  streptococcus  angina  no  benefit  could  be  ex- 
pected from  the  diphtheria  antitoxic  serum. 

Hirsh2  has  shown  that  under  pathological  conditions 
streptococci  are  by  no  means  rare  organisms  in  the  in- 
testinal canal  of  infants,  and  may  cause  a  streptococcic 
enteritis.  In  these  cases  the  organisms  are  found  in  large 
numbers  in  the  stomach  and  in  the  stools,  and  later  in 
the  course  of  the  disease  in  the  blood  and  urine  of  the 
living  child  and  in  the  internal  organs  of  the  cadaver. 

1  Fliigge's  Die  Mikroorganismen. 

2  Centralbl.  fur  Bakt.  ttnd  Parasitenk.,  Bd.  xxii.,  Nos.  14  and  15,  p.  369. 

1 3 


194  PATHOGENIC  BACTERIA. 

Liebman l  reports  two  cases  of  streptococcic  enteritis 
that  were  cerefully  studied  bacteriologically. 

Flexner,2  in  a  series  of  autopsies  upon  cases  of  death 
from  various  diseases,  found  the  bodies  invaded  by  num- 
erous micro-organisms,  causing  what  he  has  called  u  term- 
inal infections,"  and  hastening  the  fatal  issue.  Of  793 
autopsies  at  Johns  Hopkins  Hospital,  255  from  chronic 
heart  or  kidney  diseases,  or  both,  were  sufficiently  well 
studied  bacteriologically  to  meet  the  needs  of  a  statis- 
tical inquiry.  Tubercular  infection  was  not  included. 
Of  the  255  cases,  213  gave  positive  bacteriological  results. 
"The  micro-organisms  causing  the  infections,  38  in  all, 
were  the  Streptococcus  pyogenes,  16  cases;  Staphylococcus 
pyogenes  aureus,  4  cases;  Micrococcus  lanceolatus,  6  cases; 
gas  bacillus  (B.  Aerogenes  capsulatus),  three  times  alone 
and  twice  combined  with  the  Bacillus  coli  communis;  the 
gonococcus,  anthrax  bacillus,  Bacillus  proteus,  the  last 
combined  with  the  Bacillus  coli,  the  Bacillus  coli  alone,  a 
peculiar  capsulated  bacillus,  and  an  unidentified  coccus." 

It  is  interesting  to  observe  how  many  cases  were 
accompanied  by  the  streptococcus.  All  the  streptococci 
may  not  have  been  streptococcus  pyogenes,  but  for  con- 
venience in  his  statistics  they  were  regarded  by  Flexner 
as  identical. 

The  streptococcus  of  Rosenbach  is  thought  by  many 
to  be  identical  with  a  streptococcus  described  by  Fehleisen 
as  the  Streptococcus  erysipelatis  (Fig.  55).  The  two  or- 
ganisms have  much  in  common,  but  much  difference  of 
opinion  exists  upon  the  subject  of  their  identity.  It  may 
seem  unwise  to  omit  the  Streptococcus  erysipelatis  as  a 
major  topic  for  discussion,  but  the  similarity  of  the  or- 
ganism to  that  just  described  has  caused  us  to  consider 
them  in  the  same  connection. 

The  streptococci  of  erysipelas  can  be  obtained  in  almost 
pure  culture  from  the  serum  which  oozes  from  a  puncture 
made  in  the  margin  of  an  erysipelatous  patch.  They  are 

1  Ctntmlbl.  ///>•  liakt.  //>/</  l\jmsitenk.,  Bd.  xxii.,  Nos.  14  and  15,  p.  376. 
1  Journal  of  Experimental  Medicine,  vol.  i.,  No.  3,  1896. 


SUPPURA  TION.  1 95 

small  cocci,  forming  long  chains — generally  from  six  to 
ten  individuals,   but  sometimes  reaching  a  hundred  in 


FIG.  55. — Streptococcus  erysipelatis,  seen  in  a  section  through  human  skin ; 
x  500  (Frankel  and  Pfeiffer). 

number.  Occasionally  the  chains  can  be  found  collected 
in  tangled  masses.  They  can  be  cultivated  at  the  room- 
temperature,  but  grow  much  better  at  30-37°  C.  They 
are  not  particularly  sensitive  to  the  absence  of  oxygen, 
but  develop  a  little  more  rapidly  in  its  presence. 

The  erysipelas  cocci,  like  the  Streptococcus  pyogenes, 
are  not  motile,  form  no  spores,  and  are  destroyed  by  a 
low  degree  of  heat.  They  stain  well  with  aqueous  solu- 
tions of  anilin  dyes  and  also  by  Gram's  method. 

The  colonies  upon  gelatin  and  the  development  in 
gelatin  tubes,  upon  agar-agar,  and  upon  blood-serum 
are  identical  with  the  descriptions  of  the  Streptococcus 
pyogenes.  No  growth  occurs  on  potato. 

The  growth  in  bouillon  is  generally  luxuriant,  and  in 
a  short  time  causes  the  medium  to  be  filled  with  chains 
of  the  cocci.  As  the  growth  progresses  these  chains 
gather  in  clusters  and  fall  to  the  bottom  as  a  whitish 


1 96  PA  THOGENIC  BA  CTERIA . 

granular  precipitate,  above  which  the  liquid  remains 
clear. 

When  injected  into  animals  Fehleisen's  coccus  behaves 
exactly  like  the  Streptococcus  pyogenes. 

Observation  has  shown  that  dire  results  may  follow  the 
entrance  of  this  organism  into  exposed  wounds,  and  that 
it  causes  not  only  local  suppuration,  but  sometimes  a 
general  infection. 

The  empiric  experience  that  the  occasional  accidental 
infection  of  malignant  tumors  with  erysipelas  cocci  was 
followed  by  sloughing  and  subsequent  disappearance  of 
the  tumor,  suggested  inoculation  with  the  Streptococcus 
erysipelatis  as  a  therapeutic  measure.  The  dangerous 
character  of  the  remedy,  however,  caused  many  to  re- 
frain from  its  use,  for  when  one  inoculated  the  living 
erysipelas  germs  into  the  tissues  he  never  could  estimate 
the  exact  amount  of  disturbance  that  would  follow.  The 
difficulty  seems  to  have  been  overcome  by  Coley,  who 
recommends  the  toxin  instead  of  the  living  coccus  for 
injection.  A  virulent  culture  is  obtained,  inoculated 
into  small  flasks  of  slightly  acid  bouillon,  allowed  to 
grow  for  three  weeks,  then  reinoculated  with  Bacillus 
prodigiosus,  allowed  to  grow  for  ten  or  twelve  days  at 
the  room-temperature,  well  shaken  up,  poured  into  bottles 
of  about  f  3ss  capacity,  and  rendered  perfectly  sterile  by  an 
exposure  to  from  50-60°  C.  for  an  hour.  It  is  claimed 
that  the  combined  toxins  of  erysipelas  and  prodigiosus 
are  much  stronger  than  the  simple  erysipelas  toxin.  The 
best  effects  are  found  in  cases  of  sarcoma,  where  the 
toxin  causes  a  rapid  necrosis  of  the  tumor  tissue,  which 
can  be  scraped  out  with  an  appropriate  instrument. 
Numerous  cases  are  on  record  in  which  this  treatment 
has  been  most  efficacious;  but,  although  Coley  recom- 
mends it  and  Czerny  still  upholds  it,  the  majority  of  sur- 
geons have  failed  to  secure  the  desired  results. 

Recently  (1895)  considerable  attention  has  been  be- 
stowed upon  the  anti-streptococcus  scrum  of  Marmorek, 
which  is  said  to  act  specifically  upon  cases  of  strepto- 


SUPPURA  TION.  197 

coccus-infection,  both  general  and  local.  Numerous 
cases  are  upon  record  in  which  the  serum  seemed  to  exert 
a  beneficial  action. 

It  would  seem  as  if  an  antiphlogistic  serum  should 
occupy  an  important  place  in  the  future  of  medicine. 

The  serum  is  prepared  upon  the  same  plan  as  that  of 
Behring,  except  that  living  virulent  streptococci  instead 
of  the  sterile  toxin  are  injected  into  the  horse. 

BACILLUS  PYOCYANEUS. 

In  some  cases  the  pus  evacuated  from  wounds  exhibits 
a  peculiar  bluish  or  greenish  color,  from  the  presence  of 


^*Vt- 

FIG.  56. — Bacillus  pyocyaneus,  from  an  agar-agar  culture;    x   IOOO   (Itzerott 

and  Niemann). 

the  Bacillus  pyocyaneus  (Figs.  56,  57).  This  is  a  short, 
delicate  bacillus  of  small  size,  measuring  0.3  :  1-2  /«,  ac- 
cording to  Fliigge,  frequently  united  in  chains  of  four  or 
six.  It  has  round  ends,  is  actively  motile,  has  one 
terminal  flagellum,  does  not  form  spores,  and  can  exist 
with  or  without  oxygen,  though  it  is  an  almost  purely 
aerobic  organism. 

It  stains  well  with  the  ordinary  solutions,  but  does  not 
retain  the  color  by  Gram's  method. 


198  PATHOGENIC  BACTERIA. 

The  superficial  colonies  upon  gelatin  plates  form  small, 
irregular,  ill-defined  collections,  which  produce  a  fluores- 
cence of  the  neighboring 
gelatin.  The  gelatin  soft- 
ens gradually,  and  about 
five  days  elapse  before  ;'••_; 
liquefaction  is  complete. 

The    microscope    shows 
the  colonies  to  be  round,     \ 
coarsely-granulated  masses 


FIG.  57.  —  Bacillus  pyocyaneus:  colonies  upon  gelntin  (Abbott). 

with  notched  or  filamentous  borders.  They  have  a  yel- 
low-green color.  Upon  the  surface  they  form  a  delicate 
clump  with  a  smooth  surface,  finely  granular,  distinctly 
green  in  the  middle  and  pale  at  the  edges.  The  colonies 
sink  into  the  gelatin  as  the  liquefaction  progresses. 

In  gelatin  puncture-cultures  most  of  the  development 
occurs  at  the  upper  part  of  the  tube,  where  a  deep  saucer 
of  liquefaction  forms.  The  growth  slowly  descends  into 
the  medium,  and  is  the  point  of  origin  of  a  beautiful 
fluorescence.  The  bacterial  growth  sinks  to  the  bottom 
as  it  ages.  At  times  a  delicate  mycoderma  forms  on  the 
surface. 

Upon  agar-agar  the  growth  is  at  first  bright  green, 
developing  all  along  the  line  of  inoculation.  The  green 
pigment  (fluorescin)  is  soluble,  and  soon  saturates  the  cul- 
ture-medium and  makes  it  very  characteristic.  As  the 
culture  ages,  or  if  the  medium  upon  which  it  grows 
contains  much  peptone,  a  second  pigment  (pyocyanim  is 
developed,  and  the  bright  green  fades  to  a  deep  blue- 
green,  dark-blue,  or  in  some  few  cases  to  a  deep  reddish- 
brown. 

A  well-known  feature  of  the  growth  upon  fresh  agar- 
agar,  upon  which  much  stress  has  recently  been  laid  by 


SUPPURA  TION.  199 

Martin  is  the  formation  of  crystals  in  fresh  cultures. 
Crystal-formation  in  cultures  of  other  bacteria  usually 
takes  place  in  old,  partially  dried  agar-agar  cultures.  The 
bacillus  pyocyaneus,  however,  produces  crystals  in  a  few 
days  upon  fresh  media.  In  my  experience  freshly  iso- 
lated bacilli  manifest  this  capability  more  markedly  than 
those  which  have  been  for  some  time  part  of  the  labo- 
ratory stock  of  cultures,  and  subject  to  frequent  trans- 
plantation.1 

Upon  potato  a  luxuriant  greenish  or  brownish,  smeary 
layer  is  produced.  Milk  is  coagulated  and  peptonized. 

This  bacillus  is  highly  pathogenic  for  laboratory  ani- 
mals. About  i  c.cm.  of  a  fresh  bouillon  culture,  if  in- 
jected into  the  subcutaneous  tissue  of  a  guinea-pig  or  a 
rabbit,  causes  a  rapid  edema,  a  suppurative  inflammation, 
and  death  in  a  short  time  (twenty-four  hours).  Some- 
times the  animal  lives  for  a  week  or  more,  then  dies. 
There  is  a  marked  hemorrhagic  subcutaneous  edema  at 
the  seat  of  inoculation.  The  bacilli  can  be  found  in  the 
blood  and  in  most  of  the  tissues. 

When  the  dose  is  too  small  to  prove  fatal,  suppuration 
occurs  in  many  cases. 

When  sterilized  cultures  are  injected,  the  same  results 
follow,  a  relatively  larger  quantity,  of  course,  being  re- 
quired. 

Intraperitoneal  injections  cause  suppurative  peritonitis. 

The  organism  has  been  found  in  the  human  being  in  the 
pus  in  cases  of  middle-ear  disease  (often  in  pure  culture), 
panophthalmia,  bronchopneumonia,  inflammations  of  the 
nasal  fossae,  meningitis,  etc.  Escaping  from  such  local 
lesions  into  the  blood  it  sometimes  causes  nephritis. 

It  may,  however,  be  stated  that  ordinarily  the  bacillus 
is  harmless  for  human  beings,  the  above-mentioned  ex- 
amples of  pathogenic  activity  being  marked  exceptions. 

It  is  interesting  to  observe,  in  passing,  that  this  path- 
ogeny  can  be  set  aside  by  the  immunity  which  develops 
after  a  few  inoculations  with  sterilized  cultures.  These 

1  See  Centralbl.f.  Bakt.,  xxi.,  April  6,  1897,  p.  473. 


200  PATHOGENIC  BACTERIA. 

are  easily  prepared,   as  the  thermal  death-point  deter- 
mined by  Sternberg  is  56°  C. 

The  bacillus  appears  to  be  rather  common  as  a  sapro- 
phyte, and,  as  it  has  been  found  in  the  perspiration, 
probably  is  not  uncommon  upon  the  skin. 

Before  leaving  the  subject  of  suppuration  attention 
must  be  called  to  several  rather  common  bacteria  which 
may  at  times  be  the  cause  of  troublesome  suppuration. 
Among  these  are  the  pneumococcus  of  Frankel  and 
Weichselbaum,  the  typhoid  bacillus,  and  the  Bacillus 
coli  communis  (q.  v.). 

The  pneumococcus  has  not  infrequently  been  discov- 
ered most  unexpectedly  in  abscesses  of  the  brain  and 
other  deep-seated  organs,  and  seems  to  have  powerful 
chemotactic  powers.  For  a  careful  consideration  of  it 
the  reader  must  be  referred  to  the  chapter  upon  Pneumo- 
nia, where  it  is  considered  in  full. 

The  Bacillus  coli  communis,  which  is  always  present  in 
the  intestine,  seems  at  times  to  enter  the  blood-  or  lymph- 
channels  and  stimulate  suppuration,  and  numerous  cases 
are  on  record  showing  this.  The  points  most  frequently 
attacked  seem  to  be  the  bile-ducts  and  the  vermiform  ap- 
pendix, though  the  significance  of  the  organism  in  appen- 
dicitis has  no  doubt  been  overrated.  It  has  also  been  found 
in  the  kidney  in  scarlatinal  nephritis,  and  is  thought  to 
be  the  exciting  cause  of  some  cases.  It  was  originally 
described  by  Passet  as  the  Bacillus  pyogenes  fcetidiis. 
For  a  more  particular  study  of  this  organism  the  reader 
is  referred  to  the  chapter  devoted  to  its  consideration. 

The  Bacillus  typhosus  is  probably  less  frequently  a  cause 
of  suppuration  than  either  of  the  others,  yet  it  seems  to 
be  the  occasional  cause  of  the  purulent  sequelae  of  typhoid 
fever.  A  case  has  recently  been  reported  by  Flexner  in 
which  metastatic  abscesses  were  found  to  be  caused  by  it. 

The  Micrococcus  tetragcnus  has  also  been  found  in  the 
pus  of  acute  abscesses:  it  is  quite  common  in  the  cavities 
of  pulmonary  tuberculosis,  and  may  aid  in  the  destructive 
processes  involved  in  the  general  phthisical  infection. 


SUPPURA  TION.  201 

MICROCOCCUS  GONORRHCM;. 

All  authorities  now  accept  the  "gonococcus"  to  be 
the  cause  of  gonorrhea.  It  was  first  observed  in  the 
urethral  and  conjunctival  secretions  of  gonorrhea  and 
purulent  ophthalmia  by  Neisser  in  1879.  The  organisms 
are  of  hemispherical  shape,  arranged  in  pairs,  so  that 
the  inner  surfaces  are  separated  from  each  other  by  a 
narrow  interval.  Sometimes,  instead  of  pairs  of  cocci, 
fours  are  seen,  the  group  no  doubt  resulting  from  the 
division  of  a  pair. 


<%  A>^ 
jf  »  -  i 


/SO*,*.  ^*n 

-•r-  •*  *    -a?-/^'-  •-.  •" 

^»^  ^1  y      ",>N**^       ••   **flm  ^^ 

r^1  ^  V"-s» *jt-**fy- 

IJ^1  :".>* 


'*rv 


jfc 

FIG.  58. — Gonococcus  in  urethral  pus ;    x   1000  (Frankel  and  Pfeiffer). 

The  described  hemispherical  shape  is  not  exactly  cor- 
rect, for  a  good  lens  generally  shows  the  approximated 
surfaces  to  be  somewhat  concave  rather  than  flat.  The 
Germans  see  in  the  organism  a  resemblance  to  their  pop- 
ular biscuit  called  a  "semmel." 

The  gonococcus  is  small,  is  not  motile,  like  other  cocci, 
is  not  provided  with  flagella,  and  does  not  have  spores. 
It  stains  readily  with  all  the  aqueous  anilin  dyes — best 
with  rather  weak  solutions — but  not  by  Gram's  method. 
It  can  be  found  in  the  urethral  discharges  of  gonorrhea 
from  the  beginning  until  the  end  of  the  disease,  though 
in  the  later  days  its  numbers  maybe  outweighed  by  other 


202  PA  THOGENIC  BA  CTERIA . 

organisms.  Wertheim  cultivated  the  gonococcus  from  a 
case  of  chronic  urethritis  of  two  years'  standing,  and 
proved  its  virulence  by  producing  with  it  gonorrhea  in 
a  human  being.  The  organisms  are  generally  found 
within  the  pus-cells  (Fig.  58)  or  attached  to  the  surface 
of  epithelial  cells,  and  should  always  be  sought  for  as 
diagnostic  of  gonorrhea,  especially  as  urethritis  some- 
times is  caused  by  other  organisms,  as  the  Bacillus  coli 
communis1  and  the  Staphylococcus  pyogenes. 

The  cultivation  of  the  gonococcus  is  not  an  easy  task, 
but  one  which  requires  considerable  bacteriologic  skill. 
Wertheim  accomplished  it  by  diluting  a  drop  of  the  pus 
in  a  little  liquid  human  blood-serum,  then  mixing  this 
with  an  equal  part  of  melted  2  per  cent,  agar-agar  at  40° 
C.,  and  pouring  into  Petri  dishes.  As  soon  as  the  media 
became  firm  the  dishes  were  stood  in  the  incubator  at 
37°  C.,  and  in  twenty-four  hours  the  colonies  could  be 
observed.  Those  upon  the  surface  showed  a  dark  centre, 
around  which  a  delicate  granular  zone  could  be  made 
out. 

When  one  of  these  colonies  is  transferred  to  a  tube  of 
human  blood-serum  or  the  above  mixture  obliquely  co- 
agulated, isolated  little  gray  colonies  occur ;  later  these 
become  confluent  and  produce  a  delicate  smeary  layer 
upon  the  medium.  The  main  growth  is  surrounded  by 
a  thin,  veil-like  extension  which  gradually  fades  away 
into  the  medium.  A  slight  growth  occurs  upon  the 
water  of  condensation. 

Turro  says  that  the  gonococci  may  also  be  cultivated 
upon  acid  gelatin,  upon  gelatin  containing  acid  urine, 
and  also  in  acid  urine  itself,  in  which  the  gonococci  grow 
near  the  surface,  while  the  pus-cocci  which  may  be  mixed 
with  them  sink  deeper  into  the  medium.  His  work  has 
not  been  confirmed  by  other  investigators. 

Heiinan,2  who  made  an  extensive  series  of  culture-ex- 

1  V:m  der  Pluyn  and  Loag:  Centralbl.  f.  Bakt.  u.  Parasitenk.,  Bd.  xvii., 
Nos.  7,  8,  Feb.  28,  1895,  p.  233. 

2  .I/,-,/.  Record,  Dec.  19,  1886. 


SUPPURA  TION.  203 

periments,  finds  that  the  goiiococcus  grows  best  in  a  mix- 
ture of  i  part  of  pleuritic  fluid  and  2  parts  of  2  per  cent, 
agar.  Wright1  prefers  a  mixture  of  urine,  blood-serum, 
peptone,  and  agar- agar. 

It  is  ordinarily  presumed  that  gonorrhea  cannot  be 
communicated  to  animals,  but  Turro  asserts  that  the 
gonococci  when  grown  upon  acid  gelatin  readily  com- 
municate urethritis  to  dogs,  and  that  no  Icesio  continni  is 
necessary,  the  simple  introduction  of  the  organisms  into 
the  meatus  sufficing  to  produce  the  disease. 

The  injection  of  gonococci  into  the  subcutaneous  tissue 
does  not  produce  abscess. 

There  is  no  doubt  that  the  gonococcus  causes  gonor- 
rhea, as  it  has  on  several  occasions  been  intentionally 
inoculated  into  the  human  urethra  with  resulting  typical 
gonorrhea.  It  is  constantly  present  in  the  disease,  and 
very  frequently  also  in  the  sequelae — endometritis,  salpin- 
gitis,  oophoritis,  cystitis,  peritonitis,  arthritis,  conjuncti- 
vitis, endocarditis,  etc. — and,  so  far  as  can  at  present  be 
determined,  is  never  found  under  normal  conditions.  .. 

In  the  beginning  of  their  activities  the  cocci  grow  in 
the  superficial  epithelial  cells,  but  soon  penetrate  between 
the  cells  to  the  deeper  layers,  where  they  continue  their 
irritation  as  the  superficial  cells  desquamate.  Authorities 
differ  as  to  whether  the  gonococci  can  penetrate  squamous 
and  columnar  epithelium  with  equal  facility. 

The  periurethral  abscesses  that  occur  in  the  course  of 
gonorrhea  are  generally  due  to  the  Staphylococci  aureus 
and  albus,  not  directly  to  the  gonococcus. 

In  certain  of  the  remote  secondary  inflammations  the 
gonococci  disappear  after  a  time,  and  either  the  inflam- 
mation subsides  or  is  maintained  by  other  bacteria.  In 
synovitis  this  does  not  seem  to  be  true,  and  the  inflam- 
mation excited  may  last  for  months. 

As  long  as  the  gonococci  persist  the  patient  may  spread 
contagion.  It  must  be  pointed  out  that  after  apparent 
recovery  from  the  disease  the  cocci  sometimes  remain 

1  Jour,  of  the  Amer.  Med.  Assoc,,  Feb.,  1895. 


204  PATHOGENIC  BACTERIA. 

latent  in  the  urethra,  and  cause  a  relapse  if  the  patient 
partake  of  some  substance,  as  alcohol,  irritating  to  the 
mucous  membranes.  Bearing  this  in  mind,  patients 
should  not  too  soon  be  discharged  as  cured. 

The  gonococci  are  not  easily  killed,  but  withstand  dry- 
ing very  well.  Kratter  was  able  to  demonstrate  their 
presence  upon  washed  clothing  six  months  after  the  orig- 
inal soiling,  and  also  found  that  they  still  stained  well. 

Bumm  found  cocci  similar  to  the  gonococcus  in  the 
urethra,  and  points  out  that  neither  the  shape  nor  the 
position  in  the  cells  is  positively  characteristic,  but  that, 
in  addition,  there  must  be  refusal  to  stain  by  Gram's 
method  before  we  can  say  with  certainty  that  cocci  found 
in  urethral  pus  are  gonococci. 

All  of  the  urethral  inflammations  do  not  depend  upon 
the  gonococcus,  and  in  true  gonorrhea  all  of  the  inflam- 
matory symptoms  do  not  depend  upon  the  gonococcus,  as 
the  epithelial  denudation  following  the  disease  permits 
the  entrance  of  the  common  pus  cocci  of  the  urethra  into 
the  peri-urethral  tissues.  The  peri-urethral  abscesses  and 
salpingitis,  etc.,  not  infrequently  depend  upon  the  ordi- 
nary pus  cocci,  and  I  have  seen  a  case  of  gonorrhea  with 
double  orchitis  and  general  septic  infection,  with  endo- 
carditis, in  which  the  gonococci  had  no  role  in  the  sep- 
sis, which  was  caused  by  a  large  dumbbell-coccus  that 
stained  beautifully  by  Gram's  method. 

MUMPS,  OR  EPIDEMIC  PAROTITIS. 

This  epidemic,  infectious  disease  of  childhood,  charac- 
terized by  enlargement  of  the  parotid  and  submaxillary 
glands,  and  rarely  of  the  testicles,  ovaries,  and  mammae, 
has  not  been  proved  to  have  a  specific  micro-organism. 

Pasteur  thought  the  disease  due  to  bacilli  which  he 
found  in  the  blood.  Capitan  and  Charrin1  and  Olivier 
found  in  the  blood,  urine,  and  saliva  both  cocci  and  ba- 
cilli, but  their  studies  are  too  early,  and  hence  too  crude 
to  be  of  any  value. 

1  Cotnptes  Kendu  Soc.  de  Bioc.  de  Paris,  May  28,  1881. 


SUPPURA  TION.  205 

Bouchard,  Boisnet,  and  Bordas  also  found  micro-organ- 
isms in  the  blood  and  saliva. 

Netter,  Laveran,  Catrin,  Mecray,  and  Walsh  have  all 
studied  cases  and  isolated  a  diplococcus  thought  to  be 
specific.  The  organism  is  described  as  occurring  in  pairs 
and  in  fours,  sometimes  in  zooglea.  It  grows  slowly  in  the 
ordinary  media,  clouding  bouillin  in  twenty-four  hours, 
and  appearing  on  gelatin  after  forty-eight  hours  as  small 
white  punctiform  colonies  which  develop  very  slowly 
and  liquefy  some  considerable  time  after  coalescence. 
It  grows  on  potato,  and  has  a  whitish  appearance  not 
easy  to  detect.  Laveran  and  Catrin  found  the  organism 
in  67  out  of  72  cases  examined.  In  their  method  a 
few  drops  of  exudate  are  withdrawn  from  the  inflamed 
gland  with  a  hypodermic  needle,  some  of  the  negative 
results  being  due  to  the  fact  that  the  needle  withdrew  no 
exudate.  The  blood  gave  pure  cultures  in  10  out  of  15 
trials. 

Mecray  and  Walsh  report  that  by  disinfecting  the 
mouths  of  patients,  suffering  from  mumps,  with  a  satu- 
rated boric  acid  solution,  and  cleansing  Stensen's  duct, 
by  careful  massage  expressing  its  secretion,  and  then 
allowing  a  piece  of  cotton  saturated  with  a  boric  acid 
solution  to  remain  for  five  minutes  between  the  orifice  of 
the  duct  and  the  jaw,  they  were  able  to  secure  from  the 
interior  of  the  duct  upon  a  bougie  of  sterile  catgut  a 
micrococcus  identical  with  that  Laveran  had  found.  Of 
tubes  inoculated  with  the  contents  of  Stensen's  duct  6 
gave  a  mixed  growth.  All,  however,  showed  the  diplo- 
coccus. Out  of  8  carefully  made  blood  examinations,  3 
gave  pure  cultures  of  the  coccus  and  3  mixed  cultures;  2 
were  negative. 

From  Stensen's  duct  in  healthy  children  they  obtained 
the  various  oral  bacteria,  but  not  the  diplococcus  found 
in  the  cases  of  mumps.  The  experimenters  do  not  think 
it  possible  that  this  diplococcus  is  the  Staphylococcus 
epidennidis  albtis,  as  its  growth  is  slower  and  the  lique- 
faction of  gelatin  is  accomplished  only  after  a  longer 


206  PATHOGENIC  BACTERIA. 

time  than  is  required  by  the  staphylococcus.  They  did 
not  succeed  in  producing  mumps  in  animals.  In  their 
experience  a  dog  was  encountered  which  suffered  from 
swelling  of  the  parotids,  malaise,  etc.,  after  playing  with 
a  child  suffering  from  mumps. 

Concerning  the  diplococcus,  it  appeared  in  twos  and  fours ; 
rarely  in  larger  groups.  Each  was  regularly  rounded  and 
about  the  size  of  the  pus  cocci.  The  colonies  are  small, 
white,  glistening,  distinctly  denned,  regularly  circular 
spots,  at  first  discrete  and  of  slow  growth,  gradually  coa- 
lescing. The  slow  growth  is  characteristic.  In  study- 
ing pure  cultures,  some  gelatin  tubes  three  days  after  in- 
oculation were  set  aside,  no  growth  being  noted;  three 
days  later  the  small  white  colonies  became  distinctly  vis- 
ible. At  ordinary  temperatures  gelatin  is  not  liquefied 
until  ten  or  twelve  days,  and  the  liquefaction  proceeds 
slowly.  A  faint  white  streak  appears  on  potato  on  the 
third  day,  and  spreads  as  a  delicate  whitish  film.  The 
growth  upon  blood-serum  is  more  rapid  than  on  other 
media,  but  the  colony  is  not  so  distinctly  white  in  color. 
Litmus  milk  is  changed  to  pink  on  the  third  day  and  is 
coagulated.  Milk  is  thought  to  be  an  excellent  nutrient 
medium,  and  a  possible  ready  means  of  spreading  con- 
tagion. 

In  the  paper  of  Mecray  and  Walsh  no  mention  is  made 
of  the  relation  of  the  cocci  to  pus  cells  or  other  organized 
constituents  of  the  secretion  from  which  they  were 
obtained;  no  animal  inoculations  were  done  and  nothing 
is  said  about  the  reaction  to  Gram's  method  of  staining 
or  possible  motility  the  cocci  might  possess. 

Michaelis  and  Bein,1  of  Leyden's  clinic,  found  a  diplo- 
coccus (previously  observed  by  Leyden  in  the  sputum), 
which  occurred  chiefly  in  the  pus  cells.  In  severe  cases 
of  the  disease,  which  they  studied  by  culture  and  micro- 
scopic section,  the  organism  was  not  only  secured  from 
Stensen's  duct,  but  in  2  cases  from  the  pus  of  an  abscess 
(parotid  ?)  and  in  i  case  from  the  blood. 

1  Deutsche  med.  Wockenschrift,  May  13,  1897. 


SUPPURA  TION.  307 

In  spite  of  the  small  number  of  cases  studied,  they 
were  of  the  opinion  that  their  coccus  is  the  specific  one. 
It  is  about  i  p.  in  size  and  resembles  the  gonococcus, 
though  it  is  smaller.  The  cocci  generally  lie  in  the 
cells,  sometimes  8  or  10  in  one  pus  cell,  and  are  occasion- 
ally distributed  throughout  the  pus  in  long  chains  or 
strings.  They  stain  readily  with  the  usual  anilin  dyes, 
especially  with  Loftier' s  methylene-blue,  and  can  be 
decolorized  by  the  Gram  method.  They  grow  slowly 
upon  the  ordinary  media,  forming  living,  transparent, 
dew-like  points  on  agar-agar.  These  little  drops  do  not 
coalesce.  In  peptone-bouillon  they  form  white,  rather 
granular  than  flocculent  deposit,  the  bouillon  itself  re- 
maining clear.  The  growth  is  said  to  be  more  rapid  in 
strongly  than  feebly  alkaline  media.  The  cocci  are  said 
to  grow  upon  ascites-fluid  and  upon  milk,  the  latter  coag- 
ulating in  the  course  of  forty-eight  hours.  They  are 
capable  of  slight  movement.  Numerous  inoculation  ex- 
periments were  made,  only  one  animal,  a  white  mouse, 
succumbing.  Control-experiments  failed  to  disclose  the 
same  organisms  in  the  healthy  human  parotid  or  its  se- 
cretion. 

All  the  observers  agree  in  finding  in  the  secretions  of 
the  gland  and  in  the  blood  diplococci  that  grow  slowly, 
produce  small  colonies,  and  coagulate  milk.  No  one  has 
shown  their  specificity  by  inoculation,  evidence  of  course 
necessary  before  the  claim  of  real  importance  can  be 
accepted. 


II.   THE   CHRONIC   INFLAMMATORY   DISEASES. 


CHAPTER    I. 
TUBERCULOSIS. 

TUBERCULOSIS  is  one  of  the  most  dreadful  and,  un- 
fortunately, most  common  diseases  of  mankind.  It  affects 
alike  the  young  and  the  old,  the  rich  and  the  poor,  the 
male  and  the  female,  the  enlightened  and  the  savage. 
Nor  do  its  ravages  cease  with  human  beings,  for  it  is 
common  among  animals,  occurring  with  great  frequency 
among  cattle,  less  frequently  among  goats  and  hogs,  and 
sometimes,  though  rarely,  among  sheep,  horses,  dogs, 
and  cats. 

Wild  animals  under  natural  conditions  seem  to  escape 
the  disease,  but  when  caged  and  kept  in  zoological  gar- 
dens even  the  most  resistant  of  them — lions,  tigers,  etc. — 
are  said  at  times  to  succumb  to  it,  while  it  is  the  most 
common  cause  of  death  among  captive  monkeys. 

The  disease  is  not  even  limited  to  mammals,  but  occurs 
in  a  somewhat  modified  form  in  birds,  and,  it  is  said, 
even  at  times  affects  reptiles. 

It  is  not  a  disease  of  modern  times,  but  one  which  has 
persisted  through  centuries ;  and  though,  before  the  ad- 
vent of  the  microscope,  not  always  clearly  separated 
from  cancer,  it  has  not  only  left  unmistakable  signs  of 
its  existence  in  the  early  literature  of  medicine,  but  has 
also  imprinted  itself  upon  the  statute-books  of  some 
countries,  as  Naples,  where  its  ravages  were  great  and 
the  means  taken  for  its  prevention  radical. 

While  the  great  men  of  the  early  days  of  pathology 

clearly  saw  that  the  time  must  come  when  the  parasitic 
Ma 


TUBERCUL  OS  IS. 


209 


nature  of  this  disease  would  be  proved,  and  some,  as 
Klebs,  Villemin,  and  Cohnheim,  were  "within  an  ace" 
of  the  discovery,  it  remained  for  Robert  Koch  to  succeed 
in  demonstrating  and  isolating  the  specific  bacillus,  now 
so  well  known,  and  to  write  so  accurate  a  description  of 
the  organism  and  the  lesions  it  produces  as  to  render  it 
almost  unparalleled  in  medical  literature. 

The  tubercle  bacillus  (Fig.  59)  is  a  rod-shaped  organ- 


FlG.  59. — Section  of  a  peritoneal  tubercle  from  a  cow,  showing  the  tubercle 
bacilli;    x  500  (Frankel  and  Pfeiffer). 


ism  with  rounded  ends  and  a  slight  curve,  measuring 
from  1.5-3.5  p  in  length  and  from  0.2-0.5  //  in  breadth. 
It  very  commonly  occurs  in  pairs,  which  may  be  asso- 
ciated end  to  end,  but  generally  overlap  somewhat  and 
are  not  attached  to  each  other.  It  is  very  common  to 
observe  a  peculiar  beaded  appearance  in  organisms  found 
in  pus  and  sputum  (Fig.  60),  due  to  the  contraction  of 
fragmented  protoplasm  within  the  resisting  capsule  (?). 
By  some  these  fragmentations  are  thought  to  be  bacilli 
in  the  stage  of  sporulation  (see  Fig.  61).  Koch  origin- 
ally held  this  view  himself,  but  researches  have  not  been 
able  to  substantiate  the  opinion,  and  at  present  the  evi- 


14 


210  PATHOGENIC  BACTERIA. 

dences  pro  and  con  point  more  strongly  in  the  negative 
than  in  the  positive  direction. 

The  fragments  do  not  look  like  the  spores  of  any  other 
organisms.     When   spores   occur   in   the   continuity   of 


FIG.  60. — Tubercle  bacillus  in  sputum  (Frankel  and  Pfeiffer). 

bacilli,  they  are  generally  discrete  oval  refracting  bodies 
easily  recognized.  The  fragments  seen  in  the  tubercle 
bacillus  are  irregular  and  biconcave  instead  of  oval,  have 


FlG.  6l. — Tubercle  bacilli:  I,  forms  suggesting  sporulation;  2,  forms  de- 
scribed as  leaded ;  the  open  spaces  in  the  fragmented  rods  are  sometimes  mis- 
taken for  spores. 

ragged  surfaces,  and  are  without  the  refraction  peculiar 
to  the  ordinary  spore. 

The  spaces  between  the  bacillary  fragments  cannot  be 
made  to  stain  like  the  spores  of  other  species.  Finally, 


TUBERCULOSIS.  21 1 

all  known  spores  resist  heat  more  strongly  than  the  fully- 
developed  bacilli,  but  experimentation  has  shown  that 
these  degenerative  forms  are  no  more  capable  of  resist- 
ing heat  than  the  tubercle  bacilli  themselves. 

The  organism  is  not  motile,  and  does  not  possess 
flagella. 

The  tubercle  bacillus  is  peculiar  in  its  reaction  to  the 
anil  in  dyes.  It  is  rather  difficult  to  stain,  requiring  that 
the  dye  used  shall  contain  a  mordant  (Koch),  but  it  is  also 
very  tenacious  of  the  color  once  assumed,  resisting  the 
decolorizing  power  of  strong  mineral  acids  (Ehrlich). 

These  peculiarities  delayed  the  discovery  of  the  bacil- 
lus for  a  considerable  time,  but  now  that  we  are  familiar 
with  them  they  give  us  a  most  valuable  diagnostic  cha- 
racter, for  with  the  exception  of  the  bacillus  of  lepra  no 
known  bacillus  reacts  in  exactly  the  same  way. 

Koch  first  stained  the  bacillus  with  an  aqueous  solu- 
tion of  a  basic  anilin  dye  to  which  some  potassium 
hydrate  was  added,  subsequently  washing  with  water 
and  counter-staining  with  vesuvin.  Ehrlich  subsequently 
modified  Koch's  method,  showing  that  pure  anilin  was 
a  better  mordant  than  potassium  hydrate,  and  that  the 
use  of  a  strong  mineral  acid  would  remove  the  color 
from  everything  but  the  tubercle  bacillus.  This  modi- 
fication of  Koch's  method  given  us  by  Ehrlich  is  at  the 
present  time  acknowledged  to  be  the  best  method  of 
staining  the  bacillus.  Many  other  methods  have  been 
suggested,  all  of  them,  perhaps,  more  convenient  than 
Ehrlich' s,  but  none  so  good. 

As  being  that  most  frequently  performed  by  the 
physician,  we  will  first  describe  the  method  of  seeking 
the  bacillus  in  sputum. 

If  one  desires  to  be  very  exact  in  his  examination, 
it  may  be  well  to  have  the  patient  cleanse  the  mouth 
thoroughly  upon  waking  in  the  morning,  and  after  the 
first  fit  of  coughing  expectorate  into  a  clean  wide- 
mouthed  bottle.  The  object  of  this  is  to  avoid  the 
presence  of  fragments  of  food  in  the  sputum. 


212  PA  THOGENIC  BA  CTERIA . 

The  physician  will  secure  a  better  result  if  the  exam- 
ination be  made  on  the  same  day  than  if  he  wait  a  num- 
ber of  days,  because  if  the  bacilli  are  few  they  occur 
most  plentifully  in  the  small  caseous  flakes  to  be  de- 
scribed farther  on,  which  are  easily  found  at  first,  but 
which  break  up  and  become  part  of  a  granular  sediment 
that  always  forms  in  decomposed  sputum. 

The  fresh  sputum  when  held  over  a  black  surface 
generally  shows  a  number  of  grayish-yellow,  irregular, 
translucent  granules  somewhat  smaller  than  the  head  of 
a  pin.  These  consist  principally  of  the  caseous  material 
from  tuberculous  tissue,  and  are  the  most  valuable  part 
of  the  sputum  for  examination.  One  of  the  granules  is 
picked  up  with  a  pointed  match-stick  and  spread  over 
the  surface  of  a  perfectly  clean  cover-glass.  If  no  such 
fragment  can  be  found,  the  purulent  part  is  next  best  for 
examination.  The  mucus  itself  rarely  contains  bacilli 
when  free  from  scraps  of  tissue  and  pus. 

In  cases  in  which  this  ordinary  procedure  fails  to  reveal 
bacilli  whose  presence  is  strongly  indicated  by  the  clin- 
ical signs,  the  exact  method  of  searching  for  them  is  to 
partially  digest  the  sputum  with  caustic  potash,  and  then 
collect  the  solid  matter  with  a  centrifugal  apparatus.  If 
a  very  few  bacilli  are  present  in  the  sputum,  this  method 
will  often  secure  them. 

The  material  spread  upon  the  cover-glasses  should  not 
be  too  small  in  amount.  Of  course  a  massive,  thick 
layer  will  become  opaque  in  staining,  but  should  the 
layer  spread  be,  as  is  often  advised,  "  as  thin  as  possible," 
there  may  be  too  few  bacilli  upon  the  glass  to  enable  one 
to  make  a  satisfactory  diagnosis. 

As  usual,  the  material  is  allowed  to  dry  thoroughly, 
and  is  then  passed  three  times  through  the  flame  for 
purposes  of  fixation. 

Ehrlictfs  Method,  or  the  Koch-Ehrlich  Method.—  The 
cover-glasses  thus  prepared  are  floated,  smeared  side 
down,  upon,  or  immersed,  smeared  side  up,  in,  a  small 
dish  of  Ehrlich's  anilin-water  gentian-violet  solution  : 


TUBERCULOSIS.  213 

Anilin,  4, 

Saturated  alcoholic  solution  of  gentian  violet,    1 1, 
Water,  100, 

and  placed  in  an  incubator  or  a  paraffin  oven,  and  kept 
for  twenty-four  hours  at  about  the  temperature  of  the 
body.  When  removed  from  the  stain  they  are  washed 
momentarily  in  water,  and  then  alternately  in  25-33 
per  cent,  nitric  acid  and  60  per  cent,  alcohol,  until  the 
blue  color  of  the  gentian  violet  is  almost  entirely  lost. 
It  must  be  remembered  that  the  action  of  the  strong  acid 
is  a  powerful  one,  and  that  too  long  a  time  must  not  be 
allowed  for  its  application.  A  total  immersion  of  thirty 
seconds  is  quite  enough  in  most  cases.  After  final  thor- 
ough washing  in  60  per  cent,  alcohol  the  specimen  is 
counter-stained  in  a  dilute  aqueous  solution  of  Bismarck 
brown  or  vesuvin.  The  excess  of  stain  is  then  washed 
off  in  water,  and  the  specimen  is  dried  and  mounted  in 
balsam.  The  tubercle  bacilli  will  appear  of  a  fine  dark 
blue,  while  the  pus-corpuscles,  epithelial  cells,  and  other 
bacteria,  having  been  decolorized  by  the  acid,  will  be 
colored  brown  by  the  counter-stain. 

This  method,  requiring  twenty-four  hours  for  its  com- 
pletion, is  naturally  one  which  has  fallen  into  disuse  for 
practitioners  who  desire  in  the  briefest  possible  time  to 
know  simply  whether  bacilli  are  present  in  the  sputum 
or  not. 

Among  clinicians  Ziehl's  method  with  carbol-fuchsin 
has  met  with  great  favor.  After  having  been  spread, 
dried,  and  fired,  the  cover-glass  is  held  in  the  bite  of  an 
appropriate  forceps  (cover-glass  forceps),  and  the  stain1 
dropped  upon  it  from  a  pipette.  As  soon  as  the  entire 
cover-glass  is  covered  with  stain  it  is  held  over  the  flame 
of  a  spirit-lamp  or  a  Bunsen  burner  until  the  stain  begins 
to  volatilize  a  little,  as  indicated  by  a  white  vapor.  When 

1  Carbol-fuchsin  (see  p.  86) : 

Fuchsin,  I ; 

Alcohol,  10; 

5  per  cent,  phenol  in  water.  100. 


214  PA  THOGENIC  BACTERIA . 

this  is  observed,  the  heating  is  sufficient,  and  the  temper- 
ature can  be  subsequently  maintained  by  intermittent 
heating. 

If  evaporation  is  allowed  to  take  place,  a  ring  of  in- 
crustation occurs  at  the  edge  of  the  area  covered  by  the 
stain  and  prevents  the  proper  action  of  the  acid.  To 
prevent  this  more  stain  should  now  and  then  be  added. 
The  staining  is  complete  in  from  three  to  five  minutes, 
after  which  the  specimen  is  washed  off  with  water,  the 
excess  of  water  absorbed  with  paper,  and  3  per  cent, 
hydrochloric  acid  in  70  per  cent,  alcohol,  25  per  cent, 
aqueous  sulphuric,  or  33  per  cent,  aqueous  nitric  acid 
solution  dropped  upon  it  for  thirty  seconds,  or  until  the 
red  color  is  just  extinguished.  The  acid  is  washed  off 
with  water,  and  the  specimen  is  dried  and  mounted  in 
Canada  balsam.  Nothing  will  be  colored  except  the  tu- 
bercle bacilli,  which  will  appear  red. 

Gabbett  modified  the  staining  by  adding  methylene 
blue  to  the  acid  solution,  which  he  makes  according  to 
this  formula: 

Methyl  blue,  2  ; 

Sulphuric  acid,  25 ; 

Water,  75. 

In  Gabbett' s  method,  after  staining  with  carbol-fuch- 
sin  the  specimen  is  washed  with  water,  acted  upon  by 
the  methylene-blue  solution  for  exactly  thirty  seconds, 
washed  with  water  until  only  a  very  faint  blue  remains, 
dried,  and  finally  mounted  in  Canada  balsam.  By  this 
method  the  tubercle  bacilli  are  colored  red,  and  the  pus- 
corpuscles,  epithelial  cells,  and  the  unimportant  bacteria 
blue. 

The  possible  relation  that  the  number  of  bacilli  in  the 
expectoration  of  consumptives  might  bear  to  the  progress 
or  treatment  of  the  case  has  been  elaborately  investigated 
by  Nuttall.1  The  total  quantity  of  sputum  expectorated 
in  twenty-four  hours  was  caught  in  covered,  scrupulously 

1  Bull,  of  the  Johns  Hopkins  Hospital,  May  and  June,  1891,  ii.,  13. 


TUBERCULOSIS.  215 

clean  conical  glasses  and  measured  therein.  The  pro- 
portion of  muco-purulent  to  fluid  matter  was  noted. 
Depending  upon  the  degree  of  viscidity  and  number  of 
bacilli  present  in  the  sputum,  a  varying  amount  of  5  per 
cent,  caustic  potash  solution  was  added  to  it  (from  one- 
sixth  to  an  equal  volume),  and  after  the  caustic  potash 
had  rendered  the  sputum  perfectly  fluid  more  or  less  water 
was  added  to  dilute  the  mixture.  The  sputum,  having 
been  measured,  was  poured  into  a  perfectly  clean  wide- 
mouthed  bottle  containing  fine  sterilized  gravel  or  broken 
glass.  Rinsings  of  a  measured  amount  of  the  caustic  pot- 
ash solution  were  used  to  free  the  conical  glass  from  what 
matter  might  remain  and  were  added  to  the  sputum. 
The  contents  of  the  bottle  were  agitated  in  a  shaking 
machine  for  five  minutes,  and  allowed  to  stand  until  the 
caustic  potash  solution  had  had  time  to  act.  As  soon  as 
the  sputum  had  become  homogeneous  an  equal  volume 
of  water  was  added,  and  the  whole  shaken  again.  The 
sputum  thus  treated  was  of  a  pale-green  or  yellowish- 
brown  color,  and  contained  only  small  fragments  of  elas- 
tic tissue.  It  was  allowed  to  stand  two  to  four  hours, 
and  then  shaken  again  for  five  to  ten  minutes. 

By  means  of  a  burette  of  original  design  drops  of  ex- 
actly equal  size  were  secured  and  caught  upon  clean 
sterile  cover-glasses.  The  drops  were  subsequently 
spread  into  an  even  film  by  a  very  fine  platinum  wire, 
while  the  cover-glass  was  rotated  upon  a  "turn-table." 
After  spreading,  the  cover-glasses  were  laid  upon  a  level 
brass  plate  slightly  warmed  to  facilitate  drying.  After 
drying,  the  cover-glasses  were  coated  with  a  serum  film 
by  spraying,  and  the  temperature  raised  to  8o°-9O°  C.  to 
coagulate  the  serum  and  retain  the  bacteria  in  place, 
after  which  they  were  carefully  stained  with  carbol- 
fuchsin  and  decolorized  with  a  solution  of  150  parts  of 
water,  50  parts  of  alcohol,  and  20-30  drops  of  pure  sul- 
phuric acid.  Prior  to  this  the  cover-glass  was  washed  in 
three  alcohols  and  subsequently  in  water,  and  if  necessary 
in  acid  and  alcohol  again. 


2l6  PATHOGENIC  BACTERIA. 

A  special  arrangement  of  the  microscope  was  devised 
for  the  purpose,  and  the  number  of  bacilli  in  each  drop 
estimated  with  extreme  care.  The  number  varied  from 
472  to  240,000.  To  estimate  the  number  of  bacilli  in  a 
given  quantity  the  number  of  drops  to  a  cubic  centimeter 
is  multiplied  by  the  number  of  bacilli  in  the  drop,  and 
then  by  the  number  of  cubic  centimeters  to  be  estimated. 

The  method  is  an  ingenious  one,  but  a  glance  down 
the  columns  of  figures  in  the  original  article  will  be 
sufficient  to  show  that  the  counting  of  the  bacilli  is 
devoid  of  any  particular  value. 

This  is  only  to  be  expected  when  one  considers  the 
pathology  of  the  disease  and  remembers  that  accidents, 
such  as  unusually  violent  cough  one  day,  modified  by 
the  use  of  sedatives  the  next,  may  cause  wide  variations 
in  the  quality  if  not  in  the  quantity  of  the  sputum. 

When  the  tubercle  bacilli  are  to  be  sought  for  in  sections 
of  tissue,  considerable  difficulty  is  at  once  encountered, 
partly  because  of  the  thickness  of  the  section  and  partly 
because  of  the  presence  of  nuclei  which  color  intensely. 

Again,  Ehrlich's  method  must  be  recommended  as  the 
most  certain  and  best  method  of  staining  a  large  number 
of  bacilli. 

The  sections  of  tissue,  if  imbedded  in  celloidin  or  par- 
affin, should  be  freed  from  the  foreign  substances.  Like 
the  cover-glasses,  they  are  placed  in  the  stain  for  twelve 
to  twenty-four  hours  at  a  temperature  of  37°  C.  Upon 
removal  they  are  allowed  to  lie  in  water  for  about  ten 
minutes  to  wash  away  the  excess  of  stain  and  to  soften 
the  tissue,  which  often  shrinks  and  becomes  brittle.  The 
washing  in  nitric  acid  (20  per  cent.)  which  follows  may 
have  to  be  continued  for  as  long  as  two  minutes.  Thor- 
ough washing  in  60  per  cent,  alcohol  follows,  after  which 
the  sections  can  be  counter-stained,  washed,  dehydrated 
in  95  per  cent,  and  absolute  alcohol,  cleared  in  xylol, 
and  mounted  in  Canada  balsam. 

A  method  which  has  attained  great  and  deserved  praise 
is  Unna's.  It  is  as  follows :  The  sections  are  placed  in 


TUBERCUL  OS  IS.  2 1 7 

a  dish  of  twenty-four-hours-old,  newly-filtered  Ehrlich's 
solution,  and  allowed  to  remain  twelve  to  twenty-four 
hours  at  the  room-temperature  or  one  to  two  hours  in 
the  incubator.  From  the  stain  they  are  placed  in  water, 
where  they  remain  for  about  ten  minutes  to  wash.  They 
are  next  immersed  in  acid  (20  per  cent,  nitric  acid)  for 
about  two  minutes,  and  become  greenish-black.  From 
the  acid  they  are  placed  in  absolute  alcohol,  and  are 
gently  moved  to  and  fro  until  the  pale-blue  color  returns. 
They  are  then  washed  in  three  or  four  changes  of  clean 
water  until  they  become  almost  colorless,  and  are  then 
removed  to  the  slide  by  means  of  a  section-lifter.  The 
water  is  absorbed  with  filter-paper,  and  then  the  slide  is 
heated  over  a  Bunsen  burner  until  the  section  becomes 
shining,  when  it  receives  a  drop  of  xylol  balsam  and  a 
cover-glass. 

It  is  said  that  sections  stained  in  this  manner  do  not 
fade  as  quickly  as  those  stained  by  Ehrlich's  method. 

The  tubercle  bacillus  also  stains  well  by  Gram's  method, 
but  as  this  is  a  general  method  by  which  many  different 
bacteria  are  colored,  it  is  ill  adapted  for  purposes  of  differ- 
entiation, especially  when  the  prosecution  of  the  charac- 
teristic methods  is  not  more  difficult. 

So  far  as  is  known,  the  tubercle  bacillus  is  a  purely 
parasitic  organism.  It  has  never  been  found  except  in 
the  bodies  and  excretions  of  animals  affected  with  tuber- 
culosis, and  in  dusts  of  which  these  are  component  parts. 
This  purely  parasitic  nature  greatly  interferes  with  the 
isolation  of  the  organism,  which  cannot  be  grown  upon 
the  ordinary  culture-media.  Koch  first  achieved  its  arti- 
ficial cultivation  by  the  use  of  blood-serum.  When 
planted  upon  this  medium  the  bacilli  are  first  apparent 
to  the  naked  eye  in  about  two  weeks,  and  occur  in  the 
form  of  small  dry,  whitish  flakes,  not  unlike  fragments 
of  chalk.  These  slowly  increase  at  the  edges,  and  grad- 
ually form  scale-like  masses  of  small  size,  which  under 
the  microscope  are  seen  to  consist  of  tangled  masses  of 
bacilli,  many  of  which  are  in  a  condition  of  involution. 


2 1 8  PA  THOGENIC  DA  CTERIA . 

The  best  method  of  obtaining  a  culture  is  to  inoculate 
a  guinea-pig  with  tuberculous  material,  allow  an  artificial 
tuberculosis  to  develop,  kill  the  animal  after  a  couple  of 
months,  and  make  the  cultures  from  the  centre  of  one  of 
the  tuberculous  glands. 

Of  course  many  technical  difficulties  must  be  over- 
come. The  tuberculous  material  used  for  inoculation 
may  be  sputum,  injected  beneath  the  skin  by  a  hypo- 
dermic syringe.  The  animal  is  allowed  to  live  for  a 
month  or  six  weeks,  then  killed.  The  autopsy  is  per- 
formed according  to  directions  already  given.  A  large 
lymphatic  gland  with  softened  contents  or  a  nodule  in  the 
spleen  being  selected  for  the  culture,  an  incision  is  made 
into  it  with  a  sterile  knife,  or -a  rigid  sterile  platinum 
wire  is  introduced ;  some  of  the  contents  are  removed 
and  planted  upon  blood-serum.  After  receiving  the  in- 
oculated material  the  tubes  are  closed,  either  by  a  rub- 
ber cap  placed  over  the  cotton  stopper,  which  is  cut  off 
and  pushed  in,  or  by  a  rubber  cork  above  the  cotton, 
the  idea  of  this  rubber  corking  being  simply  to  prevent 
evaporation.  The  tubes  must  be  kept  in  an  incubator 
at  the  temperature  of  37-38°  C. 

Kitasato  has  published  a  method  by  which  Koch  has 
been  able  to  secure  the  tubercle  bacillus  in  pure  culture 
from  sputum.  After  carefully  cleansing  the  mouth  the 
patient  is  allowed  to  expectorate  into  a  sterile  Petri  dish. 
By  this  method  the  contaminating  bacteria  from  the 
mouth  and  the  receptacle  are  excluded,  and  the  expecto- 
rated material  is  made  to  contain  only  such  bacteria  as 
were  present  in  the  lungs.  The  material  is  carefully 
washed  a  great  many  times  in  renewed  distilled  sterile 
water  until  all  bacteria  not  enclosed  in  the  mtico-purulent 
material  are  removed  ;  it  is  then  carefully  opened  with 
sterile  instruments,  and  the  culture-medium — glycerin 
agar-agar  or  blood-serum — is  inoculated  from  the  centre. 
Kitasato  has  been  able  by  this  method  to  demonstrate 
that  many  of  the  bacilli  ordinarily  present  in  tubercular 
sputum  are  dead,  although  they  continue  to  stain  well. 


TUBERCUL  OSIS. 


219 


Kitasato's  method  of  washing  the  sputum  has  been 
modified  and  simplified  by  Czaplewski  and  Hensel.1  In 
their  studies  of  whooping-cough,  instead  of  washing  the 
flakes  in  water  in  dishes,  they  shook  them  in  peptone 
water  in  test-tubes.  The  shaking  in  the  test-tube  being 
so  much  more  thorough  than  the  washing  in  dishes,  fewer 
changes  are  necessary,  three  or  four  washings  being 
sufficient. 

In  1887,  Nocard  and  Roux  gave  a  great  impetus  to 
investigations  upon  tuberculosis  by  their  discovery  that 
the  addition  of  4-8  per  cent 
of  glycerin  to  bouillon  and 
agar-agar  made  them  suitable 
for  the  development  of  the 
bacillus,  and  that  a  much 
more  luxuriant  development 
could  be  obtained  upon  these 
media  than  upon  blood-se- 
rum. The  growth  upon  such 
* '  glycerin  agar-agar  "  (Fig. 
62)  very  much  resembles 
that  upon  blood-serum.  The 
growth  upon  bouillon  with 
4  per  cent,  of  glycerin  is 
also  luxuriant.  As  tubercle 
bacilli  require  considerable 
oxygen  for  their  proper  devel- 
opment, they  grow  only  upon 
the  surface  of  the  bouillon, 
where  a  rather  thick  myco- 
derma  forms.  The  surface- 
growth  is  rather  brittle,  and  after  a  time  gradually  sub- 
sides fragment  by  fragment. 

The  tubercle  bacillus  can  be  grown  in  gelatin  to  which 
glycerin  has  been  added,  but  as  its  development  takes 
place  only  at  37°-38°  C.,  a  temperature  at  which  gelatin  is 
always  liquid,  its  use  for  the  purpose  is  disadvantageous. 

1  Centralbl.  f.  Bakt.  u.  Parasitenk.,  xxii.,  Nos.  22  and  23,  p.  643. 


FIG.  62. — Bacillus  tuberculosis  on 
"  glycerin  agar-agar." 


220  PA  THOGENIC  BA  CTERIA. 

Pawlowski  was  able  to  cultivate  the  bacillus  upon 
potato,  but  Sander,  who  found  that  it  could  be  readily 
grown  upon  various  vegetable  compounds,  especially 
upon  acid  potato  mixed  with  glycerin,  also  found  that 
upon  such  compounds  its  virulence  was  constantly  lost. 

It  has  also  been  shown  that  the  continued  cultivation 
of  the  tubercle  bacillus  upon  such  culture-media  as 
are  appropriate  so  lessens  its  parasitic  nature  that  in  the 


FIG.  63. — Bacillus  tuberculosis :  adhesive  cover-glass  preparation  from  a  fourteen- 
day-old  blood- serum  culture;    x   loo  (Frankel  and  Pfeiffer). 

course  of  time  it  can  be  induced  to  grow  feebly  upon  the 
ordinary  agar-agar. 

It  is  really  surprising  to  note  the  extremely  simple 
compounds  in  which  the  tubercle  bacillus  can  be  grown. 
Instead  of  requiring  the  most  concentrated  albuminous 
media,  as  was  once  supposed,  Proskauer  and  Beck  have 
shown  that  the  organism  can  grow  in  non-albuminous 
media  containing  asparagin,  and  that  it  can  even  be  in- 
duced to  grow  upon  a  mixture  of  commercial  ammonium 
carbonate,  0.35  per  cent.;  primary  potassium  phosphate, 
0.15  per  cent.;  magnesium  sulphate,  0.25  per  cent.; 
glycerin,  1.5  percent.  It  was  even  found  that  tuberculin 
was  produced  in  this  inorganic  mixture. 


TUBERCULOSIS.  221 

The  tubercle  bacillus  seems  to  require  a  considerable 
amount  of  oxygen  for  its  development.  It  is  also  pecu- 
liarly sensitive  to  temperatures,  not  growing  at  a  tem- 
perature below  29°  C.  or  above  42°  C.  Temperatures 
above  75°  C.  kill  it  after  a  short  exposure. 

The  tubercle  bacillus  does  not  develop  well  in  the 
light,  and  when  its  virulence  is  to  be  maintained  should 
always  be  kept  in  the  dark.  Sunlight  kills  it  in  from 
a  few  minutes  to  several  hours,  according  to  the  thick- 
ness of  the  mass  exposed  to  its  influence. 

The  widespread  character  of  tuberculosis  at  one  time 
suggested  the  idea  that  tubercle  bacilli  were  ubiquitous 
in  the  atmosphere,  that  we  all  inhaled  them,  and  that  it 
was  only  our  vital  resistance  that  prevented  us  all  from 
becoming  its  victims. 

Cornet  must  be  given  the  credit  of  having  shown  that 
such  an  idea  is  untrue,  and  that  tubercle  bacilli  only 
exist  in  the  atmospheres  frequented  by  consumptives. 
His  experiments  were  made  by  collecting  dusts  from 
numerous  places — streets,  sidewalks,  houses,  rooms,  walls, 
etc.  Injecting  them  into  guinea-pigs,  whose  constant 
susceptibility  to  the  disease  makes  them  a  very  delicate 
reagent  for  its  detection,  Cornet  showed  the  bacilli  to  be 
present  only  in  the  dust  with  which  pulverized  sputum 
was  mixed,  and  found  such  infectious  dust  to  be  most 
common  where  the  greatest  carelessness  in  respect  to 
cleanliness  prevailed. 

Our  present  knowledge  of  the  life-history  of  the  tubercle 
bacillus,  by  showing  its  indisposition  to  multiply  outside 
the  bodies  of  animals,  the  deleterious  influence  of  sun- 
light upon  it,  the  absence  of  positive  permanent  forms, 
and  its  sensitivity  to  temperatures  beyond  a  certain  range, 
confirms  all  that  Cornet  has  pointed  out,  and  shows  us 
why  the  expectoration  of  millions  of  consumptives  has 
not  rendered  our  atmospheres  pestilential. 

As  long  as  tuberculosis  exists  among  men  or  cattle,  it 
shows  that  the  existing  hygienic  precautions  are  insuf- 


222  PATHOGENIC  BACTERIA. 

ficient.  While  not  so  radical  as  to  suggest  the  unreason- 
able isolation  of  patients  and  destruction  of  property  once 
practised  in  the  kingdom  of  Naples,  the  author  would 
favor  the  registration  of  all  tuberculous  cases  as  a  means 
of  collecting  accurate  data  concerning  their  origin,  would 
insist  upon  domestic  sterilization  and  disinfection,  and 
would  have  special  hospitals  for  as  many,  especially  of 
the  poorer  classes,  among  whom  hygienic  measures  are 
almost  always  opposed,  as  could  be  persuaded  to  occupy 
them. 

It  has  already  been  declared  the  duty  of  the  physician 
to  use  every  means  in  his  power  to  prevent  the  spread 
of  infection  in  the  households  in  his  care,  and  no  disease 
is  more  deserving  of  attention  than  this  neglected  one. 
Patients  should  cease  to  kiss  the  members  of  their  fam- 
ily and  friends ;  their  individual  knives,  forks,  spoons, 
cups,  etc.  should  be  carefully  kept  apart — secretly  if  the 
patient  be  sensitive  upon  the  subject — from  those  of  the 
family,  and  scalded  after  each  meal ;  the  napkins  and 
handkerchiefs,  as  well  as  whatever  clothing  or  bed-cloth- 
ing is  soiled  by  the  discharges,  should  be  kept  apart  from 
the  common  wash,  and  boiled ;  and  of  course  the  expec- 
toration should  be  carefully  attended  to,  received  in  a 
suitable  receptacle,  sterilized  or  disinfected,  and  never 
allowed  to  dry,  for  it  has  been  shown  that  the  tubercle 
bacillus  can  remain  vital  in  dried  sputum  for  as  long  as 
nine  months.  A  very  neat  arrangement  for  collecting 
and  disposing  of  the  expectoration  is  recommended  by 
some  boards  of  health.  It  consists  of  a  metal  case  into 
which  a  pasteboard  box  is  fitted.  When  the  box  is  to  be 
emptied  the  whole  of  the  pasteboard  portion  is  removed, 
and,  together  with  the  expectoration,  burned.  The  metal 
part  is  disinfected,  provided  with  a  new  pasteboard  box, 
and  is  again  ready  for  use.  (See  Fig.  20,  page  120.)  The 
physician  should  also  give  directions  for  disinfecting  the 
bedroom  occupied  by  a  consumptive  before  it  becomes 
the  chamber  of  a  healthy  person. 

Boards  of  health  are  now  becoming  more  and  more  in- 


TUBERCULOSIS.  223 

terested  in  tuberculosis,  and,  though  exceedingly  slow 
and  conservative  in  their  movements,  are  disseminating 
literature  among  doctors  for  distribution  to  their  patients, 
with  the  hope  of  achieving  by  volition  that  which  they 
would  otherwise  regard  as  cruel  compulsion. 

The  channels  by  which  the  tubercle  bacillus  enters  the 
organism  are  varied.  A  few  cases  are  on  record  where 
the  micro-organisms  have  passed  through  the  placenta, 
so  that  a  tuberculous  mother  was  able  to  infect  her 
unborn  child.  It  is  not  impossible  that  the  passage  of 
bacilli  in  this  manner  through  the  placenta  causes  the 
development  of  tuberculosis  in  infants  after  birth,  the 
disease  having  remained  latent  during  fetal  life,  for 
Birch-Hirschfeld  has  shown  that  fragments  of  a  fetus, 
itself  showing  no  tubercular  lesions,  but  coming  from  a 
tuberculous  woman,  were  fatal  to  guinea-pigs  into  which 
they  were  inoculated. 

The  most  frequent  channel  of  infection  is  the  respira- 
tory tract,  into  which  the  finely-pulverized  dust  of  rooms 
and  streets  enters.  Probably  all  of  us  at  some  time  in 
our  lives  inhale  living  virulent  tubercle  bacilli,  yet  not 
all  of  us  suffer  from  tuberculosis.  Personal  predisposi- 
tion seems  of  great  importance,  for  it  has  been  shown 
that  without  the  formation  of  tubercles  virulent  bacilli 
may  be  present  for  considerable  lengths  of  time  in  the 
bronchial  lymphatic  glands — the  dumping-ground  of  the 
pulmonary  phagocytes. 

In  order  that  infection  shall  occur  it  does  not  seem 
necessary  that  the  least  abrasion  or  laceration  shall  exist 
in  the  mucous  lining  of  the  respiratory  tract.  The 
tubercle  bacillus  is  a  foreign  body  of  irritating  prop- 
erties, and,  lodging  upon  a  cell,  is  soon  engulfed  in  its 
protoplasm,  or,  arrested  by  a  leucocyte,  is  dragged  off  to 
some  other  region  in  whose  narrow  passages  a  most  hos- 
tile strife  doubtless  takes  place. 

Infection  also  commonly  takes  place  through  the  gas- 
tro-intestinal  tract  by  infected  food.  At  present  an  over- 
whelming weight  of  evidence  points  to  the  presence  of 


224  PATHOGENIC  BACTERIA. 

bacilli  in  the  milk  of  cattle  affected  with  tuberculosis.  It 
does  not  seem  necessary  that  tuberculous  ulcers  shall  be 
present  in  the  udders ;  indeed,  the  bacilli  have  been 
demonstrated  in  considerable  numbers  in  milk  from 
udders  without  tubercular  lesions  discoverable  to  the 
naked  eye. 

The  meat  from  tuberculous  animals  is  less  dangerous 
than  the  milk,  because  the  meat  is  nearly  always  cooked 
before  being  eaten,  while  the  milk  is  generally  taken 
uncooked.  The  bacilli  enter  the  intestinal  lymphatics, 
sometimes  produce  lesions  immediately  beneath  the  mu- 
cous membrane,  and  lead  later  on  to  the  formation  of 
ulcers  ;  but  generally  they  first  involve  the  mesenteric 
lymphatic  glands.  The  thoracic  duct  is  sometimes  af- 
fected, and  from  such  a  lesion  it  is  easy  to  understand  the 
development  of  a  general  miliary  tuberculosis.  The  oc- 
casional absorption  of  tubercle  bacilli  by  the  lacteals,  and 
their  entrance  into  the  systemic  circulation  and  subse- 
quent deposition  in  the  brain,  bones,  joints,  etc.,  are  sup- 
posed to  explain  primary  lesions  of  these  tissues. 

Infection  is  said  also  to  take  place  occasionally  through 
the  sexual  apparatus.  In  sexual  intercourse  tubercle 
bacilli  from  tuberculous  testicles  may  be  discharged  into 
the  female  organs,  with  resulting  tuberculous  lesions. 
The  infection  in  this  way  generally  is  from  the  male  to 
the  female,  primary  tuberculosis  of  the  testicle  being 
much  more  common  than  primary  tuberculosis  of  the 
uterus  or  ovaries. 

While  most  probably  rare,  in  comparison  with  the 
preceding,  wounds  also  are  avenues  of  entrance  for  the 
tubercle  bacilli.  Anatomical  tubercles  are  not  uncom- 
mon upon  the  hands  of  anatomists  and  pathologists, 
most  of  these  growths  being  tuberculous  in  character. 
An  interesting  fact  concerning  these  dermal  lesions 
is  the  exceedingly  small  number  of  bacilli  which  they 
contain. 

The  macroscopic  lesions  of  tuberculosis  are  too  familiar 
to  require  a  description  of  any  considerable  length.  They 


v 

/* .    '  •  ^ 


Tuberculosis  of  the  lung :  the  upper  lobe  shows  advanced  cheesy  consoli- 
dation with  cavity-formation,  bronchiectasis,  and  fibroid  changes;  the  lower 
lobe  retains  its  spongy  texture,  but  is  occupied  by  numerous  miliary  tubercles. 


TUBERCULOSIS.  225 

consist  in  nodes,  nodules,  or  collections  of  agminated 
nodules,  called  tubercles,  scattered  irregularly  through 
the  tissues,  which  are  devitalized  or  disorganized  by 
their  presence.  When  tubercle  bacilli  are  introduced 
beneath  the  skin  of  a  guinea-pig,  the  animal  shows  no 
sign  of  disease  for  a  week  or  two  ;  it  then  begins  to  lose 
appetite  and  gradually  to  diminish  in  flesh  and  weight. 
Examination  at  this  time  will  show  a  nodule  at  the  point 
of  injection  and  enlargement  of  the  neighboring  lymphatic 
glands.  The  atrophy  increases,  the  animal  shows  a  febrile 
reaction,  and  at  the  end  of  a  varying  period  of  time, 
averaging  about  twelve  weeks,  dies.  Post-mortem  ex- 
amination shows  a  cluster  of  tubercles  at  the  point  of 
inoculation,  enlargement  of  lymphatic  glands  both  near 
and  remote  from  the  primary  lesion  (due  to  the  presence 
of  tubercles),  and  a  widespread  invasion  of  the  lungs, 
liver,  kidneys,  peritoneum,  and  other  organs  and  tissues, 
with  tuberculous  tissue  in  a  more  or  less  advanced  con- 
dition of  necrosis.  Sometimes  there  are  no  tubercles 
discoverable  at  the  point  of  inoculation.  There  is  no 
regularity  in  the  distribution  of  the  disease.  Tubercle 
bacilli  are  demonstrable  in  immense  numbers  in  all  the 
diseased  tissues.  The  disease  as  seen  in  the  guinea-pig  is 
more  extended  than  in  other  animals  because  of  its  greater 
susceptibility,  and  the  death  of  the  animal  is  more  rapid 
than  in  other  species  for  the  same  reason.  In  rabbits  the 
lesion  runs  a  longer  course  with  similar  lesions.  In 
bovines  and  sheep  the  infection  is  generally  first  seen 
in,  and  is  principally  confined  to,  the  alimentary  appa- 
ratus and  the  associated  organs,  though  pulmonary  dis- 
ease also  occurs.  In  man  the  disease  is  chiefly  pulmonary, 
though  gastro-intestinal  and  general  miliary  forms  are  also 
common.  The  development  of  the  lesions  in  whatever 
tissue  or  animal  always  depends  upon  the  distribution  of 
the  bacilli  by  the  lymph  or  the  blood,  and  is  first  inflam- 
matory, then  degenerative,  in  type. 

The  experiments  of  Koch,   Prudden  and  Hodenphyl, 
and  others  have  shown  that  when  dead  tubercle  bacilli 

15 


226  PATHOGENIC  BACTERIA. 

are  injected  into  the  subcutaneous  tissues  of  rabbits 
small  local  abscesses  develop  in  the  course  of  a  couple 
of  weeks,  showing  that  the  tubercle  bacilli  are  chemotac- 
tically  potent. 

While  it  is  extremely  interesting  to  observe  that  this 
chemotactic  property  exists,  it  seems  to  be  by  some  other 
irritant  that  most  of  the  lesions  of  tuberculosis  are  caused. 
When  the  dead  tubercle  bacilli,  instead  of  being  injected 
en  masse  into  the  areolar  tissue,  are  so  introduced  into 
the  body — as  by  intravenous  injection — as  to  disseminate 
themselves  or  remain  in  small  groups,  the  result  is  quite 
different,  and  much  more  closely  resembles  that  of  the 
action  of  the  living  organism. 

Batimgarten,  whose  researches  were  made  upon  minute 
tubercles  of  the  iris,  has  shown  that  the  first  manifesta- 
tion of  the  irritation  caused  by  the  bacillus  is  not  the 
attraction  of  leucocytes,  but  the  stimulation  of  the  fixed 
connective-tissue  cells  of  the  part  affected.  These  cells 
increase  in  number  by  karyokinesis,  and  form  about  the 
irritating  bacterium  a  minute  focus  which  is  the  primitive 
tubercle. 

The  leucocytes  are  of  secondary  advent,  and  are  no 
doubt  attracted  both  by  the  substance  shown  by  Prudden 
and  Hodenphyl  to  exist  in  the  bodies  of  the  dead  bacilli 
and  by  the  necrotic  changes  which  already  affect  the 
primary  cells.  For  reasons  not  understood,  the  amount 
of  chemotaxis  varies  greatly  in  different  cases.  Some- 
times the  tubercles  will  be  sufficiently  purulent  in  type 
almost  to  justify  the  name  "  tubercular  abscess;"  some- 
times there  will  be  a  marked  absence  of  cellular  ele- 
ments derived  from  the  blood. 

The  important  toxic  substance  produced  by  the  bacillus 
is  evidently  not  associated  with  chemotaxis,  for  when  the 
leucocytes  are  absent  the  necrosis  which  is  so  characteris- 
tic persists. 

The  groups  of  cells  constituting  the  primitive  tubercle 
have  scarcely  reached  microscopic  proportions  before  a 
distinct  coagulation-necrosis  is  observable.  The  proto- 


TUBERCULOSIS.  227 

plasm  of  the  cells  affected  takes  on  a  hyaline  character, 
and  seems  abnormally  viscid,  so  that  contiguous  cells 
have  a  tendency  to  become  partially  confluent.  The 
chromatin  of  their  nuclei  becomes  dissolved  in  the  nu- 
clear juice  and  gives  stained  nuclei  a  pale  but  homo- 
geneous appearance.  Sometimes  this  nuclear  change  is 
only  observed  very  late.  As  the  necrosis  advances  the 
contiguous  cells  flow  together  and  form  large  protoplas- 
mic masses — giant-cells — which  contain  as  many  nuclei 
as  there  were  component  cells.  It  may  be  that  these 
nuclei  multiply  by  karyokinesis  after  the  protoplasmic 
coalescence,  but  only  one  observer,  Baumgarten,  has 
found  signs  of  this  process  in  giant-cells.  While  these 
changes  are  in  progress  in  the  cells  of  the  primary  focus, 
the  leucocytes  may  collect  in  such  numbers  as  to  obscure 
them  and  make  themselves  appear  to  constitute  the  prim- 
itive cells.  When  the  irritant  substance  is  produced  in 
considerable  quantities,  the  most  delicate  cells  die  first ; 
and  it  is  not  infrequent  to  find  a  tubercle  rich  in  leuco- 
cytes suddenly  showing  degeneration  of  these  cells,  with 
recurring  prominence  of  the  original  epithelioid  cells. 

It  has  been  taught  by  some  that  the  giant-cells  are 
produced  by  the  union  of  the  leucocytes,  but  a  careful 
observation  of  the  role  played  by  these  cells  will  convince 
one  that  such  an  origin  for  these  monstrous  cells  must  be 
very  rare. 

Giant-cells  are  not  always  produced,  for  sometimes  the 
necrotic  changes  are  so  violent  and  widespread  as  to  con- 
vert the  whole  cellular  mass  into  a  granular  detritus  of 
unrecognizable  fragments. 

Tubercles  are  constantly  avascular,  as  would  be  ex- 
pected of  a  process  which  is  a  combination  of  progressive 
irritation  and  necrosis.  The  avascularity  may  be  a  fac- 
tor in  the  necrosis  of  the  larger  tuberculous  masses,  but 
it  plays  no  part  in  the  degeneration  of  the  smallest  tuber- 
cles, which  is  purely  toxic. 

Tubercles  may  be  developed  in  any  tissue  and  in  any 
organ.  In  whatever  situation  they  occur,  space  is  occu- 


228  PATHOGENIC  BACTERIA. 

pied  at  the  expense  of  the  tissue,  whose  component  cells 
are  pushed  aside  or  else  included  in  the  nodule.  In  mil- 
iary  tuberculosis  of  the  kidney  it  is  not  unusual  to  find  a 
tubercle  including  a  whole  glomerule,  and  resolving  its 
component  thrombosed  capillaries  and  epithelium  into 
necrotic  fragments. 

As  almost  all  tissues  contain  a  supporting  tissue-frame- 
work of  connective-tissue  fragments,  some  of  these  must 
be  embodied  in  the  new  growth.  The  fibres  which  pos- 
sess little  vitality  are  more  resistant  than  cells,  and,  after 
all  the  cells  of  a  tubercle  have  been  destroyed,  will  be 
distinctly  visible  among  the  granules,  so  that  the  tubercle 
has  a  reticulated  appearance. 

As  a  rule,  tubercles  steadily  increase  in  size  by  the  in 
vasion  of  fresh  tissue.  The  tubercle  bacillus  does  not  seem 
to  find  the  necrotic  centres  of  the  tubercles  adapted  to  its 
growth,  and  completes  its  life-cycle  with  the  tissue-cells. 
It  is  unusual  to  find  healthy-looking  bacilli  in  the  necrotic 
areas,  most  of  them  being  observed  at  the  edges  of  the 
tubercle,  where  the  nutrition  is  good.  From  such  edges 
the  bacilli  are  occasionally  picked  up  by  leucocytes  and 
transported  through  the  lymph-spaces,  until  the  phago- 
cyte falls  a  prey  to  its  prisoner,  dies,  and  sows  the  seed 
of  a  new  tubercle.  However,  for  the  spread  of  tubercle 
bacilli  from  place  to  place  phagocytes  are  not  always 
necessary,  for  the  bacilli  seem  capable  of  transportation 
by  streams  of  lymph  alone. 

Notwithstanding  the  steady  advance  which  takes  place 
in  most  observed  cases  of  tuberculosis,  and  the  thoroughly 
comprehensible  microscopic  explanation  of  it,  many  cases 
of  tuberculosis  make  quite  perfect  recoveries. 

The  periphery  of  every  tubercle  is  a  zone  of  reaction, 
with  a  marked  tendency  to  granulation  and  organization. 
If  the  vital  condition  is  such  that  through  inappro- 
priate nutriment  or  through  unusually  active  phago- 
cytosis the  activity  of  the  bacilli  is  checked  or  their 
death  is  brought  about,  this  tendency  to  cicatrization  is 
allowed  to  progress  unmolested,  and  the  necrosed  mass  is 


TUBERCULOSIS.  22Q 

soon  surrounded  with  a  zone  of  newly-formed  contracting 
fibrillar  tissue,  by  which  it  is  perfectly  isolated.  In  such 
isolated  masses  lime-salts  are  commonly  deposited.  Some- 
times this  process  is  perfected  without  the  destruction  of 
the  bacilli,  but  with  their  incarceration  and  inhibition. 
Such  a  condition  is  called  latent  tuberculosis,  and  may  at 
any  time  be  the  starting-point  of  a  new  infection  and  lead 
to  a  fatal  termination. 

In  1890,  Koch  announced  some  observations  upon  toxic 
products  of  the  tubercle  bacillus  and  their  relation  to  the 
diagnosis  and  treatment  of  tuberculosis,  which  at  once 
aroused  an  enormous  but,  unfortunately,  a  transitory 
enthusiasm. 

These  observations,  however,  are  of  capital  importance. 
Koch  observed  that  when  guinea-pigs  are  inoculated 
with  a  mixture  containing  tubercle  bacilli  the  wound 
ordinarily  heals  readily,  and  soon  all  signs  of  local  dis- 
turbance other  than  enlargement  of  the  lymphatic  glands 
of  the  neighborhood  disappear.  In  about  two  weeks  there 
occurs  at  the  point  of  inoculation  a  slight  induration  which 
develops  into  a  hard  nodule,  then  ulcerates,  and  remains 
until  the  death  of  the  animal.  If,  however,  in  the  course 
of  a  short  time  the  animals  are  reinoculated,  the  course 
of  the  process  is  altogether  changed,  for,  instead  of  heal- 
ing, the  wound  and  the  tissue  surrounding  it  assume 
a  dark  color  and  become  obviously  necrotic,  and  ulti- 
mately slough  away,  leaving  an  ulcer  which  rapidly  and 
permanently  heals  without  enlargement  of  the  lymph- 
glands. 

Having  made  this  observation  with  injected  cultures 
of  the  living  bacillus,  Koch  next  observed  that  the  same 
change  occurred  when  the  secondary  inoculation  was 
made  with  pure  cultures  of  the  dead  bacilli. 

It  was  also  observed  that  if  the  material  used  for  the 
secondary  injection  was  not  too  concentrated  and  not 
too  often  repeated  (only  every  six  to  forty-eight  hours), 
the  animals  thus  treated  improved  in  condition,  and, 
instead  of  dying  of  the  tuberculosis  induced  by  the 


230  PATHOGENIC  BACTERIA. 

primary  injection  in  from  six  to  ten  weeks,  continued 
to  live,  sometimes  (Pfuhl)  as  long  as  nineteen  weeks. 

Koch  also  discovered  that  a  50  per  cent,  glycerin 
extract  of  cultures  of  the  tubercle  bacillus  produced  the 
same  effect  as  the  dead  cultures  originally  used,  and 
gave  this  substance,  tuberculin,  to  the  scientific  world 
for  experimental  purposes,  in  the  hope  that  the  prolon- 
gation of  life  observed  in  the  guinea-pig  might  be  true 
in  the  case  of  man. 

The  active  substance  of  the  "tuberculin"  seems  to  be 
an  albuminous  derivative  insoluble  in  absolute  alcohol. 
It  is  not  a  toxalbumin. 

The  action  of  the  tuberculin  upon  the  animal  organ- 
ism is  peculiar,  but  readily  understandable.  //  does  not 
exert  the  slightest  influence  upon  the  tubercle  bacillus, 
but  acts  upon  the  living  tuberculous  tissue.  In  the 
description  of  the  tissue-changes  already  given  it  has 
been  shown  that  the  tubercle  bacillus  effects  the  coagu- 
lation-necrosis of  the  cells,  but  does  not  derive  its  nutri- 
ment from  the  dead  tissue.  As  the  cells  die  and  are 
incorporated  in  the  necrotic  mass,  the  bacilli  find  the 
conditions  of  life  unfavorable,  and  likewise  seem  to  die. 
The  active  bacilli,  therefore,  are  always  found  at  the  mar- 
gins of  the  tuberculous  tissues,  where  the  cells  are  fairly 
active.  The  necrosis  is  due  to  bacillary  poisons.  When 
tuberculin  is  injected  into  the  organism  the  result  is  to 
double  the  amount  of  poisonous  influence  upon  the  cells 
surrounding  the  bacilli,  to  destroy  their  vitality,  to  re- 
move the  favorable  conditions  of  growth  from  the  organ- 
ism, and  to  leave  it  for  a  time  checkmated. 

Virchow,  who  well  understood  the  action  of  the  tuber- 
culin, soon  showed  that  as  a  diagnostic  and  therapeutic 
agent  in  man  its  use  was  attended  with  great  danger. 
The  destroyed  tissue  was  absorbed,  and  with  it  the  bacilli 
were  likewise  absorbed  and  transported  to  new  areas, 
where  a  rapid  invasion  occurred.  Old  tuberculous  lesions 
which  had  been  encapsulated  were  softened,  broken 
down,  and  became  sources  of  dangerous  infection  to  the 


TUBERCULOSIS.  231 

individual,  so  that,  a  short  time  after  its  enthusiastic 
reception  as  a  "gift  of  the  gods,"  tuberculin  was  placed 
upon  its  proper  footing  as  a  diagnostic  agent  valuable  in 
veterinary  practice,  but  dangerous  in  human  medicine, 
except  in  cases  of  lupus  and  other  external  forms  of  the 
disease  where  the  destroyed  tissue  could  be  discharged 
from  the  surface  of  the  body. 

The  method  of  preparation  of  tuberculin  is  rather 
simple.  Small  flasks  exposing  a  considerable  surface  of 
liquid  are  filled  with  about  25  c.cm.  of  bouillon  contain- 
ing about  4  per  cent,  of  glycerin.  The  bouillon  is  prefer- 
ably made  with  calf-  instead  of  ox-meat.  When  thor- 
oughly sterile  the  surfaces  are  inoculated  with  pure 
cultures  of  the  tubercle  bacillus  and  are  stood  in  an 
incubator.  In  the  course  of  two  weeks  a  slight  surface 
growth  is  apparent,  which  in  the  course  of  time  develops 
into  a  pretty  firm  pellicle  and  gradually  subsides.  At  the 
end  of  four  or  six  weeks  development  ceases  and  the 
pellicle  sinks.  The  contents  of  a  number  of  flasks  are 
then  collected  in  an  appropriate  vessel  and  evaporated 
over  a  water-bath  to  one- tenth  their  volume,  then  filtered 
through  a  Pasteur-Chamberland  filter.  This  is  crude 
tuberculin. 

When  such  a  product  is  injected  in  doses  of  a  fraction 
of  a  cubic  centimeter  an  inflammatory  and  febrile  reac- 
tion occurs.  The  inflammation  sometimes  causes  super- 
ficial tuberculous  lesions  (lupus)  to  ulcerate  and  slough 
away,  and  for  this  reason  is  of  some  value  in  therapeutics, 
although  attended  with  the  dangers  mentioned  above. 
The  fever  is  sufficiently  characteristic  to  be  of  diagnostic 
value,  though  the  tuberculin  can  only  be  used  as  a  diag- 
nostic agent  in  practice  upon  animals. 

A  recent  important  work  upon  tuberculin  has  been 
done  by  Koch.1 

In  his  experience  the  attempts  made  to  produce  im- 
munity to  the  tubercle  bacillus  by  the  injection  into 
animals  of  attenuated  cultures  proved  failures,  because 

1  Deutsche  met?.   Wochenschrift,  1897,  No.  14. 


232  PATHOGENIC  BACTERIA. 

abscesses  invariably  followed  their  introduction,  whether 
dead  or  alive,  and  nodular  growths  in  the  lungs  were 
constant  sequelae  of  their  injection  into  the  circulation. 
In  such  nodules  the  bacilli  could  be  found  unabsorbed 
and  unaltered.  It  seemed  as  if  the  fluids  of  the  body 
could  not  effect  solution  of  the  bacteria.  The  ineffectual 
attempts  at  immunization,  with  the  results  given,  probably 
depend  upon  the  inability  of  the  tissues  to  take  up  from 
the  bacilli  whatever  immunizing  substances  they  might 
contain,  first,  because  of  the  impossibility  of  dissolving 
them,  and,  second,  because  the  irritating  powers  they 
possess  interfere  with  the  direct  action  of  normal  fluids 
and  uninjured  body-cells,  and  always  subject  the  bacteria 
to  semi-pathological  conditions. 

From  these  data,  which  he  carefully  studied  out,  Koch 
concluded  that  it  would  be  necessary  to  bring  about  some 
artificial  condition  advantageous  to  the  absorption  of  the 
bacilli,  and  for  the  purpose  tried  the  action  of  diluted 
mineral  acids  and  alkalies.  The  chemical  change  brought 
about  in  this  manner  facilitated  absorption,  but  the  ab- 
sorption of  bacilli  in  this  altered  condition  was  not  fol- 
lowed by  immunity,  probably  because  the  chemical  com- 
position of  tubercle-toxin  (or  whatever  one  may  name 
the  poisonous  products  of  the  bacillus)  was  changed  by 
the  reagents  used. 

Tuberculin,  with  which  Koch  performed  many  experi- 
ments, was  found  to  produce  immunity  only  to  tubercu- 
lin, not  to  bacillary  infection. 

Pursuing  the  idea  of  fragmenting  the  bacilli,  or  in  some 
way  treating  them  chemically  in  order  to  increase  their 
solubility,  Koch  found  that  a  10  per  cent,  sodium  hydrate 
solution  yielded  an  alkaline  extract  of  the  bacillus,  which, 
when  injected  into  animals,  produced  effects  similar  to 
those  following  the  administration  of  tuberculin,  except 
that  they  were  briefer  in  duration  and  more  constant  in 
result.  The  marked  disadvantage  of  abscess-formation 
following  the  injections,  however,  remained.  This  fluid, 
when  filtered,  possessed  the  properties  of  tuberculin. 


TUBERCULOSIS.  233 

The  mechanical  fragmentation  of  the  bacilli  had  been 
used  by  Klebs  in  the  studies  of  antiphthisin  and  tubercu- 
locidin.  Koch  now  used  it  with  advantage  in  his  studies, 
and  pulverized  living,  fresh,  virulent,  but  perfectly  dry 
bacteria  in  an  agate  mortar,  in  order  to  liberate  the  ba- 
cillary  substance  from  its  protecting  envelope  of  fatty 
acid.  In  the  trituration  only  a  very  small  quantity  of  the 
bacteria  could  be  handled  at  a  time,  and  Koch  seemed 
thoroughly  aware  of  the  risk  incurred  from  inhalation  of 
the  finely  pulverized  bacillary  mass. 

Having  reduced  the  bacilli  to  fragments,  they  were 
removed  from  the  mortar  in  distilled  water,  and  collected 
by  centrifugation,  in  a  small  glass  tube,  as  a  muddy  re- 
siduum at  the  bottom  of  an  opalescent,  clear  fluid.  For 
convenience  he  named  the  clear  fluid  TO,  the  sediment 
TR.  TO  was  found  to  contain  tuberculin.  In  order 
to  separate  the  essential  poison  of  the  bacteria  as  perfectly 
as  possible  from  the  irritating  tuberculin,  the  TR  or 
fragments  were  dried  perfectly,  triturated  once  more, 
re-collected  in  fresh  distilled  water  and  re-centrifugated. 
After  the  second  centrifugation  microscopic  examination 
showed  that  the  bacillary  fragments  had  not  been  resolved 
into  a  uniform  mass,  for  when  TO  was  subjected  to  stain- 
ing with  carbol-fuchsin  and  methyl-blue  it  was  found  to 
exhibit  a  blue  reaction,  while  in  TR  a  cloudy  violet  reac- 
tion was  obtained. 

The  addition  of  50  per  cent,  of  glycerin  had  no  effect 
upon  TO,  but  caused  a  cloudy  white  deposit  to  be  thrown 
down  from  TR.  This  last  reaction  showed  that  TR  con- 
tained fragments  of  the  bacilli  which  are  insoluble  in 
glycerin. 

Experiment  showed  that  TR  had  decided  immunizing 
powers.  Injected  into  tuberculous  animals  in  too  large 
dose  it  produced  a  reaction,  but  its  effects  were  entirely 
independent  of  the  reaction.  Koch's  aim  in  using  this 
substance  in  therapeutics  was  to  produce  immunity  in 
the  patient  without  reactions,  by  gradual  but  rapid  in- 
crease of  the  dose.  In  so  large  a  number  of  cases  did 


234  PATHOGENIC  BACTERIA. 

Koch  produce  immunity  to  tuberculosis  by  the  adminis- 
tration of  TR,  that  he  thinks  it  proved  beyond  a  doubt 
that  the  observations  are  correct. 

In  making  the  TR  preparation  Koch  advises  the  use 
of  a  fresh,  highly  virulent  culture  not  too  old.  It  must 
be  perfectly  dried  in  a  vacuum  exsiccator,  and  the  tritu- 
ration,  in  order  to  be  thorough,  should  not  be  done  upon 
more  than  100  mg.  of  the  bacilli  at  a  time.  A  satisfac- 
tory separation  of  the  TR  from  TO  is  said  only  to  occur 
when  the  perfectly  clear  TO  takes  up  at  least  50  per  cent, 
of  the  solid  substance,  as  otherwise  the  quantity  of  TO  in 
the  final  preparation  is  so  great  as  to  produce  undesirable 
reactions. 

The  fluid  is  best  preserved  by  the  addition  of  20  per 
cent,  of  glycerin,  which  does  not  injure  and  prevents 
decomposition  of  the  TR. 

The  finished  fluid  contains  10  mg.  of  solid  constituents 
to  the  c.cm.,  and  before  administration  should  be  diluted 
with  physiological  salt  solution  (not  solutions  of  carbolic 
acid).  When  administering  the  remedy  to  man  the  in- 
jections are  made  with  a  hypodermic  syringe  into  the 
tissues  of  the  back.  The  beginning  dose  is  -^-J-g-  of  a  mg., 
rapidly  increased  to  20  mg.,  the  injections  being  made 
daily. 

In  speaking  of  the  results  of  experiments  upon  guinea- 
pigs,  Koch  says: 

UI  have,  in  general,  got  the  impression  in  these  ex- 
periments that  full  immunization  sets  in  two  or  three 
weeks  after  the  use  of  large  doses.  A  cure  in  tubercu- 
lous guinea-pigs,  animals  in  which  the  disease  runs,  as 
is  well  known,  a  very  rapid  course,  may,  therefore,  take 
place  only  when  the  treatment  is  introduced  early — as 
early  as  one  or  two  weeks  after  the  infection  with  tuber- 
culosis. 

4  This  rule  avails  also  for  tuberculous  human  beings, 
whose  treatnu-nt  must  not  be  begun  too  late.  ...  A 
patient  who  has  but  a  few  months  to  live  cannot  ex- 
pect any  value  from  the  use  of  the  remedy,  and  it  will 


TUBERCULOSIS.  235 

be  of  little  value  to  treat  patients  who  suffer  chiefly  from 
secondary  infection,  especially  with  the  streptococcus, 
and  in  whom  the  septic  process  has  put  the  tuberculosis 
entirely  in  the  background." 

By  proper  administration  of  the  TR  Koch  was  able  to 
render  guinea-pigs  so  completely  immune  that  they  were 
able  to  withstand  inoculations  of  virulent  bacilli.  The 
point  of  inoculation  presents  no  changes  when  the 
remedy  is  administered,  and  the  neighboring  lymph- 
glands  are  generally  normal,  or  when  slightly  swollen 
contain  no  bacilli. 

One  very  important  objection  found  by  Trudeau  and 
Baldwin  against  commercially  prepared  TR  is  that  it  is 
possible  for  it  to  contain  unpulverized,  and  hence  live, 
virulent  tubercle  bacilli.  In  the  preparation  of  the  rem- 
edy it  will  be  remembered  that  no  antiseptic  or  germicide 
was  added  to  the  solutions,  by  which  the  effects  of  acci- 
dental failure  to  crush  every  bacillus  could  be  overcome, 
Koch  having  specially  deprecated  such  additions  as  pro- 
ducing destructive  changes  in  the  TR.  Until  this  objec- 
tion can  be  removed,  and  our  confidence  that  our  attempts 
to  cure  patients  will  not  cause  their  death  be  restored,  it 
becomes  a  question  whether  TR  can  find  a  place  in 
human  medicine  at  all,  or  must  remain  an  interesting 
scientific  laboratory  demonstration. 

Probably  the  most  interesting  use  to  which  the  TR- 
tuberculin  has  thus  far  been  put  is  found  in  the  experi- 
ments of  Fisch,1  wrho  immunized  a  horse  with  it,  hoping 
to  produce  an  antitoxin  that  might  be  useful  in  treating 
tuberculosis.  His  experiment  seems  to  have  met  with 
remarkable  success,  for  the  serum  thus  secured,  which 
he  calls  "Antiphthisic  Serum,  TR,"  is  found  to  thor- 
oughly immunize  guinea-pigs  to  tuberculosis,  to  cure 
tuberculous  guinea-pigs  in  the  early  stages  of  the  dis- 
ease, and  to  neutralize  the  effects  of  tuberculin  upon 
tuberculous  animals. 

Upon  human  beings  it  is  too  early  to  make  a  positive 

1  Jour,  of  the  Amer.  Med.  Assoc.,  Oct.  30,  1897. 


236  PA  THOGENIC  BACTERIA . 

report,  but  Fisch's  cases  have  shown  remarkable  improve- 
ment. The  subject  is  pregnant  with  interest  and  deserves 
attention. 

Hirshfelder l  claims  to  have  cured  a  large  number  of 
cases  of  tuberculosis  by  the  use  of  a  preparation  known 
as  oxyttiberculin.  It  consists  of  a  4  per  cent,  glycerin- 
bouillon  culture  of  very  virulent  tubercle  bacilli,  which 
after  being  sterilized  for  one  hour,  and  filtered,  receives 
the  addition  of  8-10  volumes  of  hydrogen  peroxid,  and  is 
then  sterilized  for  ninety-six  hours  in  a  steam  apparatus. 
During  the  sterilization  the  fluid  is  kept  in  a  glass  vessel, 
plugged  with  cotton  wool.  The  peroxid  of  hydrogen  is 
renewed  every  twelve  hours. 

From  the  fluid  obtained  in  this  way  the  excess  of  the 
peroxid  is  removed  by  alkalinization.  Before  being  em- 
ployed in  human  medicine  the  remedy  is  tested  upon 
guinea-pigs.  The  dose  may  gradually  be  increased  to  20 
c.cm.  The  theory  of  action  is  based  upon  a  claimed 
destruction  of  the  toxic  property  of  the  tuberculin  by  the 
oxidation  of  the  peroxid  of  hydrogen,  which  leaves  a 
harmless  but  potent  immunizing  substance  in  the  fluid. 

Paterson2  has  suggested,  for  the  production  of  immun- 
ity to  tuberculosis,  the  use  of  gradually  increasing  doses 
of  the  serum  of  a  fowl  immunized  to  avian  tuberculosis 
by  gradually  increased  doses  of  sterilized,  attenuated,  and 
virulent  cultures  of  the  bacillus  of  avian  tuberculosis. 
Curative  results  were  observed  in  fowls  thus  treated,  and 
in  mammals  similarly  treated,  and  the  inference  drawn 
is  that  men  treated  in  the  same  manner  can  be  similarly 
benefited.  The  dose  recommended  is  2  c.cm. 

The  theory  depends  upon  the  supposed  identity  or  near 
relationship  of  the  bacilli  of  avian  and  mammalian  tu- 
berculosis. 

Klebs  has  claimed  much  advantage  from  the  treatment 
of  tuberculosis  by  antiphthisin.  According  to  the  ex- 

1  Dfuistlu:  ntfii.  Wocluuxhrift,  1897,  No.  19,  and  Jour,  of  the  Anier.  Med. 
Assoc.,  1897. 

*  Amer.  Medico- Surg.  Bull.,  Jan.  25,  1898. 


TUBERCULOSIS.  237 

perimental  studies  of  Trudeau  and  Baldwin,  however, 
antiphthisin  is  only  much  diluted  tuberculin,  and  exerts 
no  demonstrable  influence  upon  the  tubercle  bacillus  in 
vitro,  does  not  cure  tuberculosis  in  guinea-pigs,  and 
probably  inhibits  the  growth  of  the  tubercle  bacillus 
upon  culture-media  to  which  it  has  been  added,  only  by 
its  acid  reaction. 

On  the  other  hand,  Ambler  has  used  antiphthisin  with 
excellent  results  in  the  treatment  of  human  tubercu- 
losis. 

Numerous  experimenters,  prominent  among  whom  are 
Tizzoni,  Cattani,  Bernheim,  and  Paquin,  have  experi- 
mented with  the  tubercle  bacillus  and  tuberculin,  hoping 
that  the  principles  of  serum-therapy  might  be  applicable 
to  the  disease.  Nothing  positive  has,  however,  been 
achieved.  The  first-named  observers  claim  to  have  im- 
munized guinea-pigs,  in  whose  blood  an  antitoxin  formed; 
the  last-named  thinks  the  serum  of  immunized  horses 
a  specific  for  tuberculosis.  The  field  of  experimentation 
is  an  inviting  one,  though  the  chronic  course  of  the  dis- 
ease lessens  the  certainty  with  which  the  results  can  be 
estimated. 

Babes  and  Proca,  in  an  experimental  research  upon  the 
action  of  the  antituberculous  serum,  claim  for  it  a  decided 
specific  action,  and  demonstrate  experimentally  that  ani- 
mals inoculated  with  tubercle  bacilli  and  injected  with 
the  serum  are  protected  from  the  spread  of  the  disease. 

Mafucci  and  diVestra  found  that  by  injecting  guinea- 
pigs  with  serum  from  sheep  immunized  by  injections 
first  of  dead,  then  of  living  cultures  of  tubercli  bacilli, 
although  no  cures  were  brought  about,  the  vitality  of 
the  animals  was  maintained  longer.  Unprotected  animals 
died  in  fifty  to  fifty-three  days.  Those  injected  after  in- 
fection, seventy-four  days,  and  those  injected  before  infec- 
tion, ninety-one  days. 

The  author1  made  an  elaborate  study  of  the  so-called 
antituberculin,  suggested  by  Viquerat,  and  widely  praised 

1  Jour,  of  the  Anier.  Med.  Assoc.,  Aug.  21,  1897. 


238  PATHOGENIC  BACTERIA. 

by  Paquin.  For  a  long  period,  donkeys  were  injected 
with  increasing  doses  of  tuberculin,  in  order  that  an 
antitoxin — antituberculin — might  be  generated  in  their 
blood.  Experiments  upon  guinea-pigs  showed  that  the 
serum  was  powerless  to  immunize  against  the  tubercle 
bacillus,  or  to  cure  established  tuberculosis.  The  serum, 
however,  had  the  power  of  annulling  the  effects  of  tuber- 
culin upon  tuberculous  animals.  While  a  failure  experi- 
mentally, certain  clinicians  claim  that  in  practice  it  ex- 
erts a  beneficial  action  upon  patients.  Indeed,  presuming 
that  an  antituberculin  is  formed,  it  is  but  natural  that  it 
should  do  good  in  all  cases  in  which  it  is  probable  that 
the  patient  is  poisoned  by  tuberculin  or  a  similar  product. 

Rather  nearer  the  desideratum  are  the  experiments  of 
DeSchweinitz,1  who  injected  cows  and  horses  with  increas- 
ing quantities  of  bouillon  cultures  of  a  greatly  attenuated 
tubercle  bacillus,  and  subsequently  found  that  the  serum 
possessed  the  property  of  rendering  guinea-pigs  immune 
to  the  virulent  bacilli. 

The  Bacillus  of  Fowl-tuberculosis  ( Tuberculosis  gal- 
linarum}. — The  cases  of  tuberculosis  which  occasionally 
occur  spontaneously  in  chickens,  parrots,  ducks,  and  other 
birds  were  originally  attributed  to  the  Bacillus  tuberculo- 
sis hominis,  but  the  recent  works  of  Rivolta,  Mafucci, 
Cadio,  Gilbert,  Roget,  and  others  have  shown  that,  while 
very  similar  in  many  respects  to  the  Bacillus  tuberculosis, 
the  organism  found  in  the  disease  of  birds  has  distinct 
peculiarities  which  stamp  it  a  different  variety,  but  not  a 
separate  species.  Cadio,  Gilbert,  and  Roger  succeeded  in 
infecting  fowls  by  feeding  them  upon  food  containing  tu- 
bercle bacilli,  and  keeping  them  in  cages  in  which  dust 
containing  tubercle  bacilli  was  placed.  The  infection 
was  aided  by  lowering  the  temperature  with  antipyrin 
and  lessening  vitality  by  starvation.  Morphologically, 
the  organisms  are  similar,  the  bacillus  of  fowl-tuber- 
culosis being  a  little  longer  and  more  slender  than  its 
ally. 

1  Centralbl.f.  Bakt.  und  Parasitenk.,  Sept.  15,  1897,  Bd.  xxii.,  Nos.  8  and  9. 


TUBERCULOSIS.  239 

Upon  culture-media  a  distinct  rapidity  of  growth  is 
observable,  and  we  find  that,  instead  of  growing  only 
where  glycerin  is  present,  the  Bacillus  tuberculosis  gaili- 
narum  will  grow  upon  blood-serum,  agar-agar,  and  bouil- 
lon as  ordinarily  prepared.  It  will  not  grow  upon  potato. 
The  bacillus  will  grow  at  42-43°  C.  quite  as  well  as  at 
37°  C.,  while  the  growth  of  the  tubercle  bacillus  ceases 
at  42°  C.  Moreover,  the  temperature  of  43°  C.  does  not 
attenuate  its  virulence.  The  thermal  death-point  is  70° 
C.  Upon  culture-media  it  can  retain  its  virulence  for 
two  years. 

The  growth  upon  artificial  culture-media  is  luxuriant, 
and  lacks  the  dry  quality  characteristic  of  ordinary 
tubercle-bacillus  cultures.  As  it  becomes  old  a  culture 
of  fowl-tuberculosis  turns  slightly  yellow. 

Birds  are  the  most  susceptible  animals  for  experimental 
inoculation,  the  embryos  and  young  more  so  than  the 
adults  ;  guinea-pigs  are  quite  immune.  Artificial  inocu- 
lation can  only  be  made  in  the  subcutaneous  tissue,  never 
through  the  intestine.  The  chief  seat  of  the  disease  is 
the  liver,  where  cellular  nodes,  lacking  the  central  coag- 
ulation and  the  giant-cells  of  mammalian  tuberculosis, 
and  enormously  rich  in  bacilli,  are  found.  The  disease 
never  begins  in  the  lungs,  and  the  fowls  which  are  dis- 
eased never  show  bacilli  in  the  sputum  or  the  dung. 

Rabbits  are  easily  infected,  an  abscess  forming  at  the 
seat  of  inoculation,  and  later  nodules  forming  in  the 
lung,  so  that  the  distribution  is  quite  different  from  that 
seen  in  birds. 

The  bacillus  stains  like  the  tubercle  bacillus,  but  takes 
the  stain  rather  more  easily.  The  resistance  to  acids  is 
about  the  same. 

Pseudo-tuberculosis. — Eberth,  Chantemesse,  Charrin, 
and  Roger  have  reported  certain  cases  of  so-called  pseudo- 
tuberculosis.  The  disease  occurred  spontaneously  in 
guinea-pigs,  and  was  characterized  by  the  formation  of 
cellular  nodules  in  the  liver  and  kidneys  much  resembling 
miliary  tubercles.  Cultures  made  from  them  showed  the 


240 


PATHOGENIC  BACTERIA. 


presence  of  a  small  motile  bacillus  which  could  easily  be 
stained  by  ordinary  methods  (Fig.  64).     When  introduced 


FIG.  64. — Bacillus  pseudo-tuberculosis  from  agar-agar ;    x  1000  (Itzerott  and 

Niemann). 

subcutaneously  into  guinea-pigs  the  original  disease  was 
produced. 

Pseudo-tuberculosis  seems  to  be  an  indefinite  affection 
of  which  we  have  very  little  knowledge,  and  which  is 
certainly  in  no  way  connected  with  or  related  to  true 
tuberculosis. 


CHAPTER   II. 
LEPROSY. 

LEPROSY  is  a  disease  of  great  antiquity,  and  very  early 
received  much  attention  and  study.  In  giving  the  laws 
to  Israel,  Moses  included  a  large  number  of  rules  for  its 
recognition,  the  isolation  of  the  sufferers,  the  determina- 
tion of  recovery,  and  observances  to  be  fulfilled  before 
the  convalescent  could  once  more  mingle  with  his  people. 
The  Bible  is  replete  with  accounts  of  miracles  wrought 
upon  lepers,  and  during  the  times  of  biblical  tradition  it 
must  have  been  an  exceedingly  common  and  malignant 
disease. 

At  the  present  time,  although  we  in  the  Northern 
United  States  hear  very  little  about  it,  leprosy  is  still  a 
widespread  disease.  It  exists  in  much  the  same  form  as 
two  thousand  years  ago  in  Palestine,  Syria,  Egypt,  and 
the  adjacent  countries.  It  is  exceedingly  common  in 
China,  Siam,  and  parts  of  India.  Cape  Colony  has  many 
cases.  In  Europe,  Norway,  Sweden,  and  parts  of  the 
Mediterranean  coast  furnish  a  considerable  number  of 
cases.  Certain  islands,  especially  the  Sandwich  Islands, 
are  regular  hot-beds  for  its  maintenance.  The  United 
States  is  not  exempt,  the  Gulf  coast  being  chiefly  af- 
fected. 

At  one  time  the  view  was  prevalent  that  the  disease 
was  spread  only  by  contagion,  at  another  that  it  was 
miasmatic.  At  present  the  tendency  is  to  view  it  as 
contagious  to  a  degree  rather  less  than  tuberculosis. 
Sometimes  it  is  hereditary. 

The  cause  of  leprosy  is  now  pretty  certainly  deter- 
mined to  be  the  lepra  bacillus  (Fig.  65),  which  was  dis- 

16  241 


242 


PATHOGENIC  BACTERIA. 


covered  by  Hansen,  and  subsequently  clearly  described 
by  Neisser. 

The  bacillus  is  almost  the  same  size  as  the  tubercle 
bacillus — perhaps  a  little  shorter — but  lacks  the  curve 
which  is  so  constant  in  the  latter.  It  stains  in  very 
much  the  same  way  as  the  tubercle  bacillus,  but  permits 
of  a  rather  more  rapid  penetration  of  the  stain,  so  that 


FIG.  65. — Bacillus   leprre,  seen   in  a  section   through  a  subcutaneous   node  j 
x  500  (Frankel  and  Pfeiffer). 

the  ordinary  aqueous  solutions  of  the  anilin  dyes  color 
it  quite  readily.  It  stains  well  by  Gram's  method, 
by  which  beautiful  tissue  specimens  can  be  prepared. 
The  peculiar  property  of  retaining  the  color  in  the 
presence  of  the  mineral  acids  which  characterizes  the 
tubercle  bacillus  also  characterizes  the  lepra  bacillus, 
and  the  methods  of  Ehrlich,  Gabbett,  and  Unna  can  be 
used  for  its  detection. 

Like  that  of  the  tubercle  bacillus,  its  protoplasm  often 
presents  open  spaces  or  fractures,  which  have  been  re- 


LEPROSY.  243 

garded  by  some  as  spores,  but  which  are  even  less  likely 
to  be  spores  than  the  similar  appearances  in  the  tubercle 
bacillus. 

The  organism  almost  always  occurs  singly  or  in  irreg- 
ular groups,  filaments  being  unknown.  It  is  not  motile. 

Many  experimenters  have  endeavored  to  grow  this  ba- 
cillus upon  artificially  prepared  substances,  but  in  spite 
of  modern  methods,  improved  apparatus,  and  refined 
media,  few  claim  to  have  met  with  success. 

Bordoni-Uffredozzi  was  able  to  grow  upon  a  blood-serum- 
glycerin  mixture  a  bacillus  which  partook  of  the  staining 
peculiarities  of  the  lepra  bacillus  as  it  appears  in  the 
tissues,  but  differed  very  much  from  it  in  its  morphology. 
After  numerous  generations  this  bacillus  was  induced  to 
grow  upon  ordinary  culture-media.  It  commonly  pre- 
sented a  club-like  form,  which  was  regarded  by  Baum- 
garten  as  an  involution  appearance.  Frankel  points  out 
that  the  bacillus  of  Bordoni  is  possessed  of  none  of  the 
essential  characters  of  the  lepra  bacillus  except  its  stain- 
ing. 

Czaplewski l  offers  a  confirmation  of  the  work  of  Bor- 
doni-Uffredozzi, together  with  a  description  of  a  bacillus 
supposed  to  be  the  lepra  bacillus,  which  he  succeeded  in 
cultivating  from  the  nasal  secretions  of  a  leper. 

The  bacillus  was  first  isolated  upon  a  culture-medium 
consisting  of  glycerinized  serum  without  the  addition  of 
salt,  pepton,  or  sugar.  The  mixture  was  placed  in  flat 
dishes,  coagulated  by  heat,  and  sterilized  by  the  inter- 
mittent method. 

The  secretion,  rich  in  lepra  bacilli,  was  taken  up  with 
a  platinum  wire  and  inoculated  upon  the  culture-medium 
by  a  series  of  linear  strokes.  The  dishes  (Petri  dishes 
were  used  for  the  experiment)  were  securely  closed  with 
paraffin  and  stood  in  the  incubating-oven  at  37°  C. 

Upon  the  surface  of  the  medium  there  grew  numerous 
colonies  of  staphylococcus  aureus,  the  bacillus  of  Fried- 

1  Cenlralbl.  f.  Bakt.  und  Parasitenk.,  Jan.  31,  1898,  vol.  xxiii.,  Nos.  3  and 
4,  p.  97. 


244  PATHOGENIC  BACTERIA. 

lander  and  a  number  of  colonies  consisting  of  fine,  slender, 
often  somewhat  nodose  bacilli  about  the  size  and  form  of 
the  lepra  bacillus. 

These  colonies  were  grayish-yellow,  humped  in  the 
middle,  1-2  mm.  in  diameter,  irregularly  rounded,  and 
irregular  at  the  edges.  They  could  be  inverted  entire 
with  the  platinum  wire  and  were  excavated  on  the  under 
side.  The  consistence  was  crumbly. 

When  a  transfer  was  made  from  one  of  these  colonies 
to  fresh  media,  in  a  few  days  the  growth  became  apparent 
and  assumed  a  band-like  form,  with  a  plateau-like  eleva- 
tion in  the  center. 

The  bacillus  thus  isolated  grew  with  moderate  rapidity 
upon  all  the  ordinary  culture-media  except  potato.  Upon 
blood-serum  the  growth  was  more  luxuriant  and  fluid 
than  upon  the  solid  media.  Upon  coagulated  serum  the 
growth  was  rather  dry  and  elevated,  and  was  frequently 
so  loosely  attached  to  the  surface  of  the  medium  as  to 
be  readily  lifted  up  by  the  platinum  wire. 

The  growth  was  especially  good  upon  sheep's  blood- 
serum  witli  the  addition  of  5  per  cent,  of  glycerin.  The 
growth  upon  the  L,6ffler-mixture  was  excellent. 

Upon  agar-agar  the  growth  is  not  so  good  as  upon 
blood-serum  ;  it  is  more  luxuriant  iipon  glycerin  agar- 
agar  than  upon  plain  agar-agar;  it  is  grayish  and  flatter 
upon  agar-agar  than  upon  blood-serum.  The  growth 
never  extends  to  the  water  of  condensation  to  form  a 
floating  layer,  as  does  that  of  the  tubercle  bacillus. 

The  colonies  that  form  upon  agar-agar  are  much  like 
those  described  by  Bordoni-Uffredozzi,  and  appear  as  iso- 
lated, grayish,  rounded  flakes,  thicker  in  the  center  than 
at  the  edges,  and  characterized  by  an  irregular  serrated 
border  from  which  a  fine  irregular  network  extends  upon 
the  medium.  These  projections  consist  of  bundles  of  the 
bacilli. 

Upon  gelatin  the  bacillus  develops  well  after  it  has 
grown  artificially  for  a  number  of  generations.  Upon 
the  surface  of  gelatin  the  growth  is,  in  general,  similar 


LEPROSY.  345 

to  that  upon  agar-agar.  In  puncture-cultures  most  of  the 
growth  is  on  the  surface  in  the  form  of  a  whitish,  or 
grayish,  or  yellowish  folded  layer.  In  the  depths  of  the 
gelatin  the  development  occurs  as  a  granular  rather  thick 
column.  The  medium  is  not  liquefied. 

Bouillon  is  not  clouded;  no  superficial  growth  occurs. 
The  vegetation  occurs  only  at  the  bottom  of  the  tube  in 
the  form  of  a  powdery  sediment. 

Czaplewski  found  that  the  bacillus  stained  well  with 
Loftier' s  methylen-blue,  and  with  the  aqueous  solutions 
of  the  anilin  dyes.  It  also  stains  by  Gram's  method,  and 
has  the  same  resisting  power  to  the  decolorizing  action 
of  mineral  acids  and  alcohol  as  the  lepra  bacillus  as  seen 
in  tissue.  The  young  bacilli  color  homogeneously,  but 
older  ones  are  invariably  granular.  They  are  usually 
pointed  at  the  ends  when  young,  but  may  be  rounded  or 
knobbed  when  older.  The  more  rapidly  the  bacillus 
grows,  the  longer  and  more  slender  it  appears. 

All  attempts  to  infect  the  lower  animals  with  leprosy, 
either  by  the  purulent  matter  or  solid  tissue  from  lepers, 
or  by  inoculating  them  with  the  supposed  specific  bacilli 
that  have  been  isolated,  have  failed. 

Ducrey  seems  to  have  cultivated  the  lepra  bacillus  in 
grape-sugar,  agar,  and  in  bouillon  "in  vacua"  His 
results  need  confirmation.  Very  few  instances  are  re- 
corded in  which  actual  inoculation  has  produced  leprosy 
in  either  men  or  animals.  Arning  was  able  to  secure 
permission  to  experiment  upon  a  condemned  criminal  in 
the  Sandwich  Islands.  The  man  was  of  a  family  entirely 
free  from  disease.  Arning  introduced  beneath  his  skin 
fragments  of  tissue  freshly  excised  from  a  lepra  nodule, 
and  kept  the  man  under  observation.  In  the  course  of 
some  months  typical  lesions  began  to  develop  at  the 
points  of  inoculation  and  spread  gradually,  ending  in 
general  lepra  in  the  course  of  about  five  years. 

Melcher  and  Artmann  introduced  fragments  of  lepra 
nodules  into  the  anterior  chambers  of  the  eyes  of  rabbits, 
and  observed  the  death  of  the  animals  after  some  months 


246  PATHOGENIC  BACTERIA, 

with  typical  lepra  lesions  of  all  the  viscera,  especially 
the  cecum. 

While  the  lepra  bacillus  has  much  in  common  with  the 
tubercle  bacillus,  there  is  not  the  slightest  evidence  of 
any  real  identity.  It  has  already  been  shown  that  lepra 
bacilli  do  not  grow  upon  artificial  media,  and  that  they 
cannot  be  readily  transmitted  by  inoculation.  The  fol- 
lowing description  will  show  that  the  relation  of  the 
bacilli  to  the  lesions  is  entirely  different  from  that  of 
the  tubercle  bacilli  to  the  tubercles. 

Like  the  Bacillus  tuberculosis,  the  Bacillus  leprae  proba- 
bly only  occurs  in  places  frequented  by  persons  suffering 
from  the  disease.  That  individuals  are  infected  by  the 
latter  less  readily  than  by  the  former  bacilli  probably 
depends  upon  the  fact  that  leprous  infection  seems  to 
take  place  most  commonly  by  the  entrance  of  the  organ- 
isms into  the  individual  through  cracks  or  fissures  in 
the  skin,  while  the  tuberculous  infection  occurs  through 
the  more  accessible  respiratory  and  digestive  apparatus. 
Once  established  in  the  body,  the  bacillus  by  its  growth 
produces  chronic  inflammatory  nodes — the  analogues  of 
tubercles. 

The  nodes  of  lepra  consist  of  various  kinds  of  cells 
and  of  fibres.  Unlike  the  tubercles,  the  lepra  nodes  are 
vascular,  and  much  of  the  embryonal  tissue  completes 
its  formative  function  by  the  production  of  fibres.  The 
bacilli  are  not  distributed  through  the  nodes  like  tubercle 
bacilli,  but  are  found  in  groups  enclosed  within  the  proto- 
plasm of  certain  large  cells — the  "  lepra  cells."  These 
cells  seem  to  be  overgrown  and  partly  degenerated  lym- 
phoid  cells.  Sometimes  they  are  anuclear,  sometimes 
they  contain  several  nuclei  (giant-cells). 

Lepra  nodules  do  not  degenerate  like  tubercles,  and 
the  formation  of  ulcers,  which  constitutes  a  large  part  of 
the  disease,  seems  largely  due  to  the  action  of  external 
agencies  upon  the  feebly  vital  pathological  tissue,  which 
is  unable  to  recover  itself  when  injured. 

According  to  the  recent  studies  of  Johnston  and  Jamie- 


LEPROSY.  247 

son,1  the  bacteriological  diagnosis  of  nodular  leprosy  can 
be  made  by  spreading  the  serum  obtained  by  scraping  a 
leprous  nodule  upon  a  cover-glass,  drying,  fixing,  and 
staining  with  carbol-fuchsin  and  Gabbet's  solution  as  for 
the  tubercle  bacillus.  In  such  preparations  the  bacilli 
are  present  in  enormous  numbers,  thus  forming  a  marked 
contrast  to  the  tubercular  skin  diseases,  in  which  very  few 
can  be  found. 

In  that  form  known  as  anesthetic  leprosy,  nodules  form 
upon  the  peripheral  nerves,  and  by  connective-tissue 
formation,  as  well  as  the  entrance  of  the  bacilli  into  the 
nerve-sheaths,  cause  irritation,  then  degeneration,  of  the 
nerves.  The  anesthesia  which  follows  these  peripheral 
nervous  lesions  is  one  of  the  conditions  predisposing  to 
the  formation  of  ulcers,  etc.  by  allowing  injuries  to  occur 
without  detection  and  to  progress  without  observation. 
The  ulcerations  and  occasional  loss  of  phalanges  that 
follow  these  lesions  occur,  probably,  in  the  same  manner 
as  in  syringomyelia. 

The  disease  advances,  having  first  manifested  itself 
upon  the  face,  extensor  surfaces,  elbows,  and  knees,  to  the 
lymphatics  and  the  internal  viscera.  Death  ultimately 
occurs  from  exhaustion,  if  not  from  the  frequent  inter- 
current  affections  to  which  the  conditions  predispose. 

1  Montreal  Med.  Journal,  Jan.,  1897. 


CHAPTER  HI. 
GLANDERS. 

GLANDERS  is  an  infectious  mycotic  disease  which,  very 
fortunately,  is  almost  confined  to  the  lower  animals.  Only 
occasionally  does  it  secure  a  victim  from  hostlers,  drovers, 
soldiers,  and  bacteriologists,  whose  frequent  association 
with  and  experimentation  upon  animals  bring  them  in 
frequent  contact  with  those  which  are  diseased.  Of  all 
the  infectious  diseases  studied  by  scientists,  none  has 
caused  the  havoc  which  glanders  has  wrought.  Several 
men  of  prominence  have  succumbed  to  accidental  in- 
fection. 

Glanders  was  first  known  to  us  as  a  disease  of  the  horse 
and  ass  characterized  by  the  occurrence  of  discrete,  clean- 
ly-cut ulcers  upon  the  mucous  membrane  of  the  nose. 
These  ulcers  are  formed  by  the  breaking  down  of  nodules 
which  can  be  detected  upon  the  diseased  membranes,  and 
show  no  tendency  to  recover,  but  slowly  spread  and  dis- 
charge a  virulent  pus.  The  edges  of  the  ulcers  are  in- 
durated and  elevated,  the  surfaces  often  smooth.  The 
disease  does  not  progress  to  any  great  extent  before  the 
submaxillary  lymphatic  glands  begin  to  enlarge.  Later 
on  these  glands  form  large  lobulated  masses,  which  may 
soften,  open,  and  become  discharging  ulcers.  The  lungs 
may  also  become  infected  by  inspiration  of  the  infectious 
material,  and  contain  small  foci  not  unlike  tubercles  in 
appearance.  The  animals  ultimately  die  of  exhaustion. 

In  1882,  shortly  after  the  discovery  of  the  tubercle 
bacillus,  Loffler  and  Schiitz  discovered  in  the  discharges 
and  tissues  of  this  disease  the  specific  micro-organism, 
the  glanders  bacillus  (Bacillus  mallei;  Fig.  66),  which  is 
its  cause. 

2iS 


GLANDERS.  249 

The  glanders  bacillus  is  somewhat  shorter  and  dis- 
tinctly thicker  than  the  tubercle  bacillus.  It  has  rounded 
ends,  and  it  generally  occurs  singly,  though  upon  blood- 


FlG.  66. — Bacillus  mallei,   from  a  culture  upon  glycerin  agar-agar;    x    IOOO 
(Frankel  and  Pfeiffer). 

serum,  and  especially  upon  potato,  several  joined  indi- 
viduals may  be  found.     Long  threads  are  never  formed. 

The  bacillus  is  non-motile.  Various  observers  have 
claimed  the  discovery  of  spores,  but  although  in  the 
interior  of  the  bacilli  there  have  been  observed  irregular 
spaces  like  the  similar  spaces  in  the  continuity  of  the 
tubercle  bacillus  not  colored  by  the  stains,  they  have 
not  yet  been  definitely  proven  to  be  spores.  (The  ob- 
servation of  Loffler  that  the  bacilli  can  be  cultivated 
after  being  kept  in  a  dry  state  for  three  months  ma_k£sjt_ 
appear  as  if  some  permanent  form  (spore)  occurs.^  No 
flagella  have  been  demonstrated  upon  the  bacillus. 

Like  the  tubercle  bacillus,  the  glanders  bacillus  does 
not  seem  to  find  conditions  outside  the  animal  body  suit- 
able for  its  existence,  and  probably  does  not  occur  except 
as  a  parasite. 

The  organism  only  grows  between  25°  and  42°  C.,  and 
generally  grows  very  slowly,  so  that  attempts  at  its  isola- 


250  PATHOGENIC  BACTERIA. 

tion  and  cultivation  by  the  usual  plate  method  are  apt  to 
fail,  because  the  numerous  other  organisms  in  the  material 
grow  much  more  rapidly. 

The  best  method  of  isolation  seems  to  be  the  use  of  an 
animal  reagent.  It  has  been  said  that  glanders  princi- 
pally affects  horses  and  asses.  Recent  observations,  how- 
ever, have  shown  the  goat,  cat,  hog  (slightly),  field-mouse, 
wood-mouse,  marmot,  rabbit,  guinea-pig,  and  hedgehog 
all  to  be  susceptible  animals.  Cattle,  house-mice,  white 
mice,  and  rats  are  immune. 

The  guinea-pig,  being  a  highly  susceptible  as  well 
as  a  readily  procurable  animal,  naturally  becomes  the 
reagent  for  the  detection  and  isolation  of  the  bacillus. 
When  a  subcutaneous  inoculation  of  some  glanders  pus 
is  made,  the  disease  can  be  observed  in  guinea-pigs 
by  a  tumefaction  in  from  four  to  five  days.  Somewhat 
later  this  tumefaction  changes  to  a  caseous  nodule,  which 
ruptures  and  leaves  a  chronic  ulcer,  with  irregular  mar- 
gins. The  lymph-glands  speedily  become  involved,  and 
in  a  month  to  five  weeks  signs  of  general  infection  are 
present.  The  lymph-glands  suppurate,  the  testicles  un- 
dergo the  same  process,  and  still  later  the  joints  exhibit 
a  suppurative  arthritis  containing  the  bacilli.  The  ani- 
mal finally  dies  of  exhaustion.  In  guinea-pigs  no  nasal 
ulcers  form.  In  field-mice,  which  are  even  more  suscepti- 
ble, the  disease  is  much  more  rapid.  No  local  lesions 
are  visible.  In  two  or  three  days  the  animal  seems  un- 
well, the  breathing  is  hurried,  it  sits  still  with  closed 
eyes,  and  without  any  other  preliminaries  tumbles  over 
on  its  side,  dead. 

From  the  tissues  of  the  inoculated  animals  the  pure 
cultures  are  most  easily  made.  Perhaps  the  best  places 
to  secure  the  culture  are  from  softened  nodes  which  have 
not  ruptured  or  from  the  suppurating  joints.  Strauss 
has,  however,  given  us  a  method  which  is  of  great  use, 
because  of  the  short  time  required.  The  material  sus- 
pected to  contain  the  glanders  bacillus  is  injected  into 
the  peritoneal  cavity  of  a  male  guinea-pig.  In  three  or 


GLANDERS.  251 

four  days  the  disease  becomes  established.  The  testicles 
enlarge  a  little ;  the  skin  over  them  becomes  red  and 
shining.  The  testicles  themselves  begin  to  suppurate, 
and  often  discharge  through  the  skin.  The  animal  dies 
in  about  two  weeks.  If  such  an  animal  be  killed  and  its 
testicles  examined,  the  tunica  vaginalis  testis  will  be 
found  to  contain  pus,  and  sometimes  to  be  partially  ob- 
literated by  inflammatory  exudation.  The  bacilli  are  pres- 
ent in  this  pus,  and  can  be  secured  from  it  in  pure  cultures. 

The  value  of  Strauss' s  method  has,  however,  been  less- 
ened by  the  discovery  by  Kutcher,1  that  a  new  bacillus, 
which  he  has  classified  among  the  pseudo-tubercle  ba- 
cilli, produces  a  similiar  testicular  swelling  when  injected 
into  the  abdominal  cavity. 

The  purulent  discharges  from  the  noses  of  horses 
and  from  other  lesions  of  large  animals  generally  con- 
tain very  few  bacilli,  so  that  their  detection  by  the 
use  of  the  guinea-pig  inoculation  is  made  much  more 
simple. 

The  bacillus  is  an  aerobic  organism,  and  can  be  grown 
in  bouillon,  upon  agar-agar,  better  upon  glycerin  agar- 
agar,  very  well  upon  blood-serum,  and  quite  character- 
istically upon  potato.  It  grows  in  gelatin,  but  this  is 
not  an  appropriate  medium,  because  the  bacillus  develops 
best  at  temperatures  at  which  the  gelatin  is  liquid. 

Upon  4  per  cent,  glycerin  agar-agar  plates  the  colonies 
appear  upon  the  second  day  as  pale-yellow  or  whitish, 
shining  round  dots.  Under  the  microscope  they  appear 
as  brownish-yellow,  thick  granular  masses  with  sharp 
borders. 

The  culture  upon  agar-agar  and  glycerin  agar-agar 
occurs  as  a  moist,  shining  layer  not  possessed  of  distinct 
peculiarities.  Upon  blood-serum  the  growth  is  rather 
characteristic.  The  colonies  along  the  line  of  inoculation 
first  develop  as  circumscribed,  clear,  transparent  drops, 
which  later  become  confluent  and  form  a  transparent 
layer  unaccompanied  by  liquefaction. 

1  Zeitschrift  fur  Hygiene,  Bd.  xxi.,  Heft  i.,  Dec.  6,  1895. 


252  PATHOGENIC  BACTERIA. 

The  most  characteristic  growth  is  upon  potato.  It 
first  appears  in  about  forty-eight  hours  as  a  transparent, 
honey-like,  yellowish  layer,  developing  only  at  incuba- 
tion-temperature and  soon  becoming  reddish-brown.  As 
this  brown  color  of  the  colony  develops,  the  potato  for 
a  considerable  distance  around  it  becomes  greenish- 
brown.  (See  Frontispiece.}  No  other  known  organism 
produces  the  same  appearance  upon  potato. 

In  litmus  milk  the  growth  of  the  glanders  bacillus  is 
associated  with  the  production  of  an  acid  that  reddens 
the  reagent,  with  the  formation  of  a  firm  coagulum  and 
the  subsequent  separation  from  it  of  a  clear  reddish 
whey. 

The  organism  loses  its  virulence  if  cultivated  for  many 
generations  upon  artificial  media. 

The  bacillus  is  killed  in  five  minutes  by  exposure  to 
55°  C. 

That  this  bacillus  is  the  cause  of  glanders  there  is  no 
room  to  doubt.  Loffler  and  Schiitz  have  succeeded  by 
the  inoculation  of  horses  and  asses  in  producing  the 
well-known  disease. 

The  organisms  when  in  cultures  can  be  stained  with 
the  watery  anilin-dye  solutions,  but  are  difficult  to  stain 
in  tissues.  They  do  not  stain  by  Gram's  method. 

The  chief  difficulty  in  staining  the  bacillus  in  tissues 
is  the  readiness  with  which  it  gives  up  the  stain  in  the 
presence  of  decolorizing  agents.  Loffler  at  first  accom- 
plished the  staining  by  allowing  the  sections  to  lie  for 
some  time  (five  minutes)  in  the  alkaline  methylene-blue 
solution,  then  transferring  them  to  a  solution  of  sulphuric 
and  oxalic  acids — 

Concentrated  sulphuric  acid,  2  drops ; 

5  per  cent,  oxalic-acid  solution,  i  drop  ; 

Distilled  water,  10  c.  cm. 

for  five  seconds,  then  transferring  to  absolute  alcohol, 
xylol,  etc.  The  bacilli  appear  dark  blue  upon  a  paler 
ground.  This  method  gives  very  good  results,  but  has 


GLANDERS,  253 

been  largely  superseded  by  the  use  of  Kiihne's  carbol- 
methylene  blue : 

Methylene  blue,  1.5 

Alcohol,  10. 

5  per  cent,  aqueous  phenol  solution,         100. 

Kiihne's  method  of  staining  is  to  place  the  section  in  the 
stain  for  about  half  an  hour,  wash  in  water,  decolorize 
carefully  in  hydrochloric  acid  (10  drops  to  500  c.cm.  of 
water),  immerse  at  once  in  a  solution  of  lithium  carbonate 
(8  drops  of  a  saturated  solution  of  lithium  carbonate  in  10 
c.cm. of  water),  place  in  a  bath  of  distilled  water  for  a  few 
minutes,  dip  into  absolute  alcohol  colored  with  a  little 
methylene  blue,  dehydrate  in  anilin  oil  containing  a 
little  methylene  blue  in  solution,  wash  in  pure  anilin 
oil,  not  colored,  then  in  a  light  ethereal  oil,  clear  in 
xylol,  and  mount  in  balsam. 

When  stained  in  sections  of  tissue  the  bacilli  are 
found  to  occupy  the  interior  of  small  inflammatory  zones 
not  unlike  tubercles  in  appearance.  These  nodules  can 
be  seen  with  the  naked  eye  scattered  through  the  livers, 
kidneys,  and  spleens  of  animals  dead  of  experimental 
glanders.  The  nodules  consist  principally  of  leucocytes, 
but  also  contain  numerous  epithelioid  cells.  As  is  the  case 
with  tubercles,  the  centres  of  the  nodules  are  prone  to 
degenerate,  soften,  and  also  to  suppurate.  The  retro- 
gressive processes  upon  exposed  surfaces,  where  the  break- 
ing down  of  the  nodules  allows  their  contents  to  escape, 
are  the  sources  of  the  typical  ulcerations.  At  times  the 
process  is  progressive,  and  some  of  the  lesions  heal  by 
the  formation  of  a  stellate  scar. 

Baumgarten  regarded  the  origin  and  course  of  the  his- 
tological  lesions  of  glanders  to  be  much  like  those  of  the 
tubercle.  In  his  studies  epithelioid  cells  first  accumulated, 
and  were  followed  by  leucocytes.  Tedeschi  was  not  able 
to  confirm  the  results  of  Baumgarten' s  work,  but  found  the 
primary  change  to  be  due  to  a  necrosis  of  the  affected 
tissue  followed  by  an  invasion  of  leucocytes.  The  recent 


254  PATHOGENIC  BACTERIA. 

researches  of  J.  H.  Wright l  are  in  accord  with  those  of 
Tedeschi  rather  than  with  those  of  Baumgarten,  for 
Wright  observed  first  a  marked  degenerative  effect  upon 
the  tissue,  and  then  an  inflammatory  exudation  amount- 
ing in  some  cases  to  actual  suppuration. 

As  has  been  mentioned,  cultures  of  the  bacillus  lose 
their  virulence  more  or  less  after  four  or  five  generations 
in  artificial  media.  While  this  is  true,  attempts  to  atten- 
uate fresh  cultures  by  heat,  etc.  have  so  far  failed. 

Leo  has  pointed  out  that  white  rats,  which  are  immune 
to  the  disease,  may  be  made  susceptible  by  feeding  with 
phloridzin  and  causing  a  glycosuria. 

Kalning,  Preusse,  Pearson,  and  others  have  pre- 
pared a  substance,  "mallein,"  from  cultures  of  the 
bacillus,  and  suggested  its  employment  for  diagnostic 
purposes.  It  seems  to  be  quite  useful  in  veterinary 
medicine,  the  reaction  occasioned  by  its  injection  being 
similar  to  that  caused  by  the  injection  of  tuberculin  in 
tuberculous  patients.  The  manufacture  of  mallein  is 
not  attended  with  great  difficulty.  The  bacilli  are  grown 
in  glycerin  bouillon  for  several  weeks,  killed  by  heat,  the 
culture  filtered  through  porcelain  and  evaporated  to  one- 
tenth  of  its  volume.  It  has  also  been  prepared  from 
potato  cultures,  which  are  said  to  produce  a  stronger 
toxin.  A  febrile  reaction  of  more  than  1.5°  C.  following 
the  injection  is  said  to  be  specific  of  the  disease.  Babes 
has  asserted  that  the  injection  of  this  toxic  product  into 
susceptible  animals  will  protect  them  from  the  disease. 

Various  experiments  have  been  made  with  curative 
objects  in  view.  Certain  observers  claim  to  have  seen 
good  results  follow  the  injection  of  mallein  in  repeated 
small  doses.  Others,  as  Chenot  and  Picq,  find  the  blood- 
serum  from  immune  animals  like  the  ox  to  be  curative 
when  injected  into  infected  guinea-pigs. 

1  Jour,  of  Exp.  Med.t  vol.  i.,  No.  4,  p.  577. 


CHAPTER    IV. 

SYPHILIS. 

ALTHOUGH  syphilis  is  almost  as  well  known  as  it  is 
widespread,  we  have  not  yet  discovered  for  it  a  definite 
specific  cause.  Whether  it  is  due  to  a  protozoan  par- 
asite, or  whether  it  is  due  to  a  bacterium,  the  future 
must  decide.  Numerous  claims  have  been  made  by  those 
whose  studies  have  revealed  organisms  of  one  kind  or 
another  in  syphilitic  tissues,  but  no  one  has  yet  suc- 
ceeded either  in  isolating,  cultivating,  or  successfully  in- 
oculating them. 

In  1884  and  1885,  Lustgarten  published  a  method  for 
the  staining  of  bacilli  which  he  had  found  in  syphilitic 
tissues  and  assumed  to  be  the  cause  of  the  disease.  The 
staining,  which  is  very  complicated,  requires  that  the 
sections  of  tissue  be  stained  in  Ehrlich's  anilin-water 
gentian-violet  solution  for  twelve  to  twenty-four  hours  at 
the  temperature  of  the  room,  or  for  two  hours  at  40°  C. ; 
washed  for  a  few  minutes  in  absolute  alcohol ;  then  im- 
mersed for  about  ten  seconds  in  a  i^  per  cent,  perman- 
ganate-of-potassium  solution,  after  which  they  are  placed 
in  an  aqueous  solution  of  sulphurous  acid  for  one  to  two 
seconds,  thoroughly  washed  in  water,  run  through  alco- 
hol and  oil  of  cloves,  and  finally  mounted  in  Canada 
balsam  dissolved  in  xylol. 

If  the  bacilli  are  supposed  to  be  present  in  pus  or  dis- 
charges from  syphilitic  lesions,  the  cover-glasses  spread 
with  the  material  are  stained  in  the  same  manner,  except 
that  for  the  first  washing  distilled  water  instead  of  abso- 
lute alcohol  is  used. 

This  method  undergoes  a  modification  in  the  hands  of 
De  Giacomi,  who  prefers  to  stain  the  cover-glasses  in  hot 

255 


256  PATHOGENIC  BACTERIA. 

anilin-water-fuchsin  solution  for  a  few  moments,  sections 
in  the  same  solution  cold  for  twenty-four  hours ;  then 
immerse  them  first  in  a  weak,  then  in  a  strong,  solution 
of  chlorid  of  iron.  The  cover-glasses  are  washed  in 
water,  sections  in  alcohol,  and  subsequently  passed 
through  the  usual  reagents  for  dehydration  and  clearing. 


% 


FIG.  67. — Bacillus  of  syphilis  (Lustgarten),  from  a  condyloma;  x  1000  (Itzerott 
and  Niemann). 

In  some  syphilitic  tissues  these  methods  suffice  to  de- 
fine distinct  bacilli  with  a  remarkable  similarity  to  the 
tubercle  bacillus.  The  organism  is  about  the  same  size 
as  the  tubercle  bacillus,  and  even  more  frequently  curved, 
but  often  presents  a  club-like  enlargement  of  one 
end  (involution-form?).  The  bacilli  very  frequently 
occur  singly,  though  more  often  in  groups,  and  never  lie 
free,  but  are  always  enclosed  in  cells.  These  bacilli  are 
not  always  found  in  syphilitic  lesions,  nor  is  their  dem- 
onstration easy  under  the  most  favorable  circumstances. 
Lustgarten  emphasizes  particularly  that  they  are  only 
demonstrable  after  the  most  painstaking  technical  pro- 
cedures. 

The  probability  of  the  specificity  of  this  organism  was 
considerably  lessened  by  the  observation  by  Matterstock, 
Travel,  and  Alvarez  that  in  preputial  smegma,  and  also 


SYPHILIS.  257 

in  vulvar  smegma  from  healthy  individuals,  a  similar 
organism,  identical  both  in  morphology  and  staining 
peculiarities,  could  be  demonstrated.  Of  course  the  oc- 
currence of  Lustgarten's  bacillus  in  the  internal  organs 
could  not  but  argue  against  the  probability  of  its  identity 
with  the  smegma  bacillus  ;  but  Lustgarten  himself  pointed 
out  that  the  bacilli  of  both  tuberculosis  and  leprosy  stain 
by  his  method,  and  thus  gave  Baumgarten  the  right  to 
suggest  that  the  few  cases  well  adapted  for  the  demon- 
stration of  the  Lustgarten  bacilli  might  be  cases  of  mixed 
infection  of  tuberculosis  and  syphilis. 

The  most  recent  research  upon  the  bacteriology  of 
syphilis  is  that  of  van  Niessen,1  who  claims  to  have  cul- 
tivated a  syphilis  bacillus  from  the  blood  of  a  few  cases. 
Blood  secured  from  a  deep  puncture  at  the  end  of  a 
thoroughly  disinfected  finger  is  caught  in  a  sterile  glass, 
diluted  with  an  equal  quantity  of  distilled  water  and 
kept  for  from  ten  to  fourteen  days  at  a  temperature  of 
io°-20°  R.  (i3°-i5°  C.).  Very  often  the  blood  of  syphi- 
litics  is  found  subject  to  accidental  contamination  by 
various  well-known  bacteria.  When  this  is  not  the  case, 
however,  the  serum  remains  almost  perfectly  clear  and 
contains  a  large  number  of  bacilli — syphilis  bacilli.  The 
bacillus  can  be  transplanted  to  bouillon,  in  which  it  grows 
with  the  production  of  grayish-white  shreds  and  floating 
flocculi,  some  of  which  are  suspended  in  the  liquid,  while 
others  form  a  membrane  upon  the  surface. 

When  transplanted  to  obliquely  solidified  gelatin  and 
kept  at  room  temperature,  in  the  course  of  forty-eight 
hours  a  very  fine,  grayish-white,  thready  mass  like 
cloudy  streaks,  and  having  a  peculiar  reflecting  surface, 
can  be  seen.  Under  a  lens  this  is  seen  to  consist  of  lines 
of  threads  which  sometimes  seem  to  penetrate  into  the 
depths  of  the  gelatin.  After  a  time  a  layer  is  formed 
upon  the  surface  of  the  medium.  Some  liquefaction  of  the 
medium  occurs  and  causes  the  growth  to  slide  down  upon 

1  Centralbl.  f.  Bakt.  und  Parasitenk.,  Bd.  xxiii.,  No.  2,  Jan.  19,  1898,  p.  49; 
No.  344,  Jan.  31,  1898,  p.  97;  and  No.  546,  Feb.  II,  1898,  p.  177. 
17 


258  PATHOGENIC  BACTERIA. 

itself  so  as  to  assume  the  form  of  a  fragment  of  a  tape- 
worm. Upon  agar-agar  after  the  lapse  of  two  days  the 
growth  consists  of  a  central  pellicle  along  the  line  of  in- 
oculation, with  little  sprouts  projecting  in  all  directions 
from  the  edges.  The  growth  is  grayish,  with  an  occa- 
sional yellowish  tinge. 

Punctures  in  agar-agar  were  unsuccessful,  but  in  gela- 
tin the  appearance  of  the  growth  is  similar  to  that  of 
the  cholera  spirillum. 

The  bacillus  also  grows  upon  potato  in  the  form  of  an 
elevated  layer  of  exactly  the  same  color  as  the  potato. 
In  the  course  of  time  the  entire  potato  becomes  colored 
a  dark  gray.  It  also  grows  in  milk,  urine,  serum,  and 
water. 

The  colonies  of  this  bacillus  are  quite  characteristic, 
but  so  varied  in  appearance  as  to  make  one  suspect  that 
the  plate  upon  which  which  they  grow  is  contaminated 
with  various  other  species  of  bacteria.  In  general,  the 
colonies  may  be  said  to  appear  slowly  as  transparent 
whitish  drops,  which  become  grayish  and  later  yellow- 
ish, and  finally  brownish  in  color.  The  gelatin  about 
them  presents  concentric,  wave-like  rings,  depending 
upon  the  liquefaction  of  the  medicine. 

When  the  growth  is  more  rapid  and  occurs  at  higher 
temperatures  bundles  of  threads,  somewhat  resembling 
the  early  stages  of  a  mould,  are  observed.  Examining 
microscopically,  one  finds  in  the  slowly  growing  colonies 
a  surrounding  zone  of  small  centrifugally  arranged  fine 
threads  or  hairs  extending  in  all  directions,  with  one  or 
two  exceptionally  long  bundles  extending  beyond  the 
others  and  beyond  the  limits  of  the  colony.  The  long 
threads  are  never  found  to  divide.  Many  of  the  colonies 
are  highly  suggestive  of  those  of  anthrax. 

The  bacillus  is  motile  in  very  slight  degree.  It  forms 
spores.  It  is,  in  general,  about  the  size  of  the  tubercle 
bacillus. 

The  vegetation  of  the  organism  is  said  to  be  peculiar 
in  that  the  bacillary  stage  is  of  short  duration  and  soon 


SYPHILIS.  259 

gives  place  to  the  formation  of  septate,  V-shaped,  and 
branched  forms.  It  seems  to  be  normally  a  strepto-ba- 
cillus  in  its  early  stages,  but  eventually  becomes  very 
pleomorphous,  varying  in  appearance  from  a  chain  of 
oval  cocci  to  the  hypha  of  the  moulds.  There  seems  to 
be  nothing  peculiar  about  the  staining-capacity  of  the 
bacillus.  It  stains  with  the  ordinary  solutions  of  the 
anilin  dyes,  retains  the  stain  of  Gram's  method,  and  is 
decolorized  by  mineral  acids. 

Dohle 1  succeeded  in  staining  certain  protoplasmic 
bodies  in  the  tissues  in  syphilis,  which  resembled  the 
actively  motile  protoplasmic  bodies  which  he  had  pre- 
viously encountered  in  the  discharges.  They  were  for 
the  most  part  round  or  oval,  sometimes  with  irregular 
outlines,  and  were  provided  with  flagella.  The  staining 
took  place  in  a  mixture  of  hematoxylon  and  carbol-fuch- 
sin,  subsequently  treated  with  iodin  or  chromatin,  and 
washed  in  alcohol. 

Convinced  that  these  bodies  were  the  cause  of  syphilis, 
he  excised  small  fragments  from  gummata  and  other 
syphilitic  tissues,  and  placed  them  beneath  the  skin  of 
guinea-pigs,  which  subsequently  fell  ill  with  a  chronic 
marasums  which  ultimately  caused  death. 

In  the  inoculation  experiments  of  van  Niessen  there 
were  observed  as  evidences  of  the  specificity  of  the 
organism  discovered  by  him:  (i)  abortion  in  pregnant 
female  rabbits;  (2)  extra-genital  primary  lesions  on  the 
ears  of  inoculated  rabbits  in  the  form  of  nodes;  (3)  sec- 
ondary ulcer  and  tumor  formations,  and  irregular  lesions, 
such  as  occasional  thrombosis  and  pneumonia. 

1  Munch,  med.   Wochenschrift,  1897,  No.  43. 


CHAPTER   V. 
ACTINOMYCOSIS. 

IN  1845,  Langenbeck  discovered  that  the  specific  dis- 
ease of  cattle  known  as  actinomycosis  could  be  com- 
municated to  man.  His  observations,  however,  were  not 
given  to  the  world  until  1878,  one  year  after  Bellinger 
had  discovered  the  cause  of  the  disease  in  animals. 


FIG.  68. — Actinomyces  bovis,  from  the  tongue  of  a  calf;    x  500  (Frankel  and 

Pfeiffer). 

Actinomycosis  is  a  disease  almost  peculiar  to  the  bovine 
animals,  though  sometimes  occurring  in  hogs,  horses, 
men,  and  other  animals. 

The  first  manifestations  of  the  disease  are  usually  found 
either  about  the  jaw  or  in  the  tongue,  in  either  of  which 

260 


A  CTINOMYCOSIS.  261 

localities  there  are  produced  considerable  enlargements 
which  are  sometimes  dense  and  fibrous  (wooden  tongue) 
and  sometimes  suppurative.  In  sections  of  these  nodular 
formations  small  yellowish  granules  surrounded  by  some 
pus  can  be  found.  These  granules  when  viewed  beneath 
the  microscope  exhibit  a  peculiar  rosette-like  body — the 
ray-fungus  or  actinomyces. 

The  fungus  is  of  sufficient  size  to  be  detected  by  the 
naked  eye.  It  can  be  colored,  in  sections  of  tissue,  by 
the  use  of  Gram's  method,  or  better  by  Weigert's  fibrin 
stain.  Tissues  pre-stained  with  carmin,  then  stained  by 
Weigert's  method,  give  beautiful  pictures. 

The  entire  fungus-mass  consists  of  several  distinct 
zones  embracing  entirely  different  elements.  At  the 
centre  of  the  mass  there  is  found  a  granular  substance 
containing  numerous  bodies  resembling  micrococci.  Ex- 
tending from  this  centre  into  the  neighboring  tissue  is  a 
radiating,  apparently  branched,  thickly-tangled  mass  of 
mycelial  threads.  These  threads  seem  to  terminate  in 
a  zone  of  conspicuous  club-shaped  radiating'forms  which 
give  the  colonies  the  rosette-like  appearance.  The  cells 
of  the  tissues  affected  and  a  larger  or  smaller  collection 
of  leucocytes  form  the  surrounding  resisting  tissue-zone. 

The  degree  of  chemotactic  influence  exerted  by  the 
organism  seems  to  depend  partly  upon  the  tissue  affected 
and  partly  upon  the  individuality  of  the  animal.  When 
the  animal  is  but  slightly  susceptible,  and  when  the 
tongue  is  the  part  affected,  the  disease  is  characterized 
by  the  production  of  enlargement  due  to  the  formation 
of  cicatricial  tissue.  If,  on  the  other  hand,  the  animal 
is  highly  susceptible  or  the  jaw  is  affected,  the  chief 
symptom  is  suppuration,  with  the  formation  of  cavities 
communicating  by  sinuses. 

Before  the  nature  of  the  affection  was  understood  it 
was  confounded  with  various  diseases  of  the  bones,  prin- 
cipally with  osteosarcoma. 

From  the  tissues  primarily  affected  the  disease  spreads 
to  the  lymphatic  glands,  and  not  infrequently  to  the 


262  PATHOGENIC  BACTERIA. 

lungs.  Israel  has  pointed  out  certain  cases  of  human 
actinomycosis  beginning  in  the  peribronchial  tissues, 
probably  from  inhalation  of  the  fungi. 

The  occurrence  of  three  distinct  elements  as  compo- 
nents of  the  rays  served  to  class  this  organism  among 
the  pleomorphous  bacteria  in  the  genus  Cladothrix, 
where  it  has  remained  undisturbed  for  at  least  a  decade. 
Recent  researches  have,  however,  changed  the  view  held 
by  some  bacteriologists  in  regard  to  the  actinomyces,  and 
caused  them  to  regard  the  organism  as  a  bacillus.  If  it 
be  a  bacillus,  the  central  zone  of  granular  cocci-like 
elements  is  to  be  regarded  as  consisting  of  individuals 
in  process  of  rapid  t  division  and  spore(?)-formation,  the 
mycelial  zone  as  consisting  of  perfect  individuals,  and 
the  peripheral  zone,  with  the  rosette-like,  club-shaped 
elements,  as  consisting  of  individuals  partly  degener- 
ated through  the  activity  of  the  cells  and  tissue-juices 
(involution-forms). 

Jones  is  of  the  opinion  that  the  disease,  if  not  inden- 
tical  with,  is  closely  allied  to,  tuberculosis,  and  that  the 
occasional  branched  forms  of  tubercle  bacilli  prove  the 
tendency  of  the  individual  bacillus  to  form  a  reticulum. 

When  the  mycelial  threads  are  carefully  examined,  the 
branchings,  which  appear  distinct  upon  hasty  inspection, 
are  found  to  be  more  the  effect  of  a  peculiar  relation 
which  the  threads  bear  to  one  another  than  actual  bifur- 
cations, so  that  it  must  be  regarded  as  very  questionable 
whether  these  threads  ever  so  divide. 

The  organism  may  be  grown  upon  artificial  culture- 
media,  as  has  been  proven  by  Israel  and  Wolff. 

Upon  agar-agar  or  glycerin  agar-agar  it  forms  trans- 
lucent colonies,  about  the  size  of  a  pin's  head,  of  firm, 
almost  cartilaginous,  consistence.  These  colonies  consist 
of  bacillary  individuals,  sometimes  seemingly  branched. 
In  bouillon  similar  dense  globular  organisms  can  be 
grown.  The  blood-serum  colonies,  which  grow  simi- 
larly to  the  agar-agar  colonies,  are  rather  more  luxuri- 
ant, and  slowly  liquefy  the  medium. 


ACTINOMYCOSIS.  263 

When  the  actinomyces  are  grown  upon  artificial  media 
their  virulence  is  retained  for  a  considerable  length  of 
time.  If  introduced  into  the  abdominal  cavities  of  rab- 
bits, there  are  produced  in  the  peritoneum,  mesentery, 
and  omentum  typical  nodules  containing  the  actinomyces 
rays. 

The  organism  can  also  be  grown  in  raw  eggs,  into 
which  it  is  carefully  introduced  through  a  small  opening 
made  under  aseptic  precautions.  In  the  egg  the  organism 
forms  peculiar  long  mycelial  threads  quite  unlike  the  short 
forms  developing  upon  agar-agar. 

The  characteristic  rosettes  which  are  constantly  found 
in  the  tissues  are  never  seen  in  artificial  cultures. 

The  exact  manner  by  which  the  organism  enters  the 
body  is  unknown.  In  some  cases  it  may  be  by  direct 
inoculation  with  pus,  but  there  is  reason  to  believe  that 
the  organism  occurs  in  nature  as  a  saprophyte,  or  as 
an  epiphyte  upon  the  hulls  of  certain  grains,  especially 
barley.  Woodhead  records  a  case  where  a  primary  me- 
diastinal  actinomycosis  in  the  human  subject  was  sup- 
posed to  be  traced  to  perforation  of  the  posterior  pharyn- 
geal  wall  by  a  barley  spikelet  swallowed  by  the  patient. 

Cases  of  actinomycosis  are  fortunately  of  rare  occur- 
rence in  human  medicine,  and  do  not  always  occur  in 
those  brought  in  contact  with  the  lower  animals.  The 
fungi  may  enter  the  organism  through  the  mouth  and 
pharynx,  through  the  respiratory  tract,  through  the  di- 
gestive tract,  or  through  wounds. 

The  invasion  has  been  known  to  take  place  at  the  roots 
of  carious  teeth,  and  is  more  liable  to  occur  in  the  lower 
than  in  the  upper  jaw.  Israel  reported  a  case  in  which 
the  primary  lesion  seemed  to  occur  external  to  the  bone 
of  the  lower  jaw,  as  a  tumor  about  the  size  of  a  cherry, 
with  an  external  opening.  In  two  cases  of  the  disease 
observed  by  Murphy  of  Chicago  both  began  with  tooth- 
ache and  swelling  of  the  jaw. 

When  inhaled,  the  organisms  gain  entrance  to  the 
deeper  portions  of  the  lung,  and  bring  about  a  suppura- 


264 


PATHOGENIC  BACTERIA. 


tive  bronchopneumonia  with  adhesive  inflammation  of 
the  contiguous  pleura.  After  the  formation  of  the  pleu- 
ritic adhesions  the  disease  may  penetrate  the  newly 
formed  tissue,  extend  to  the  chest- wall,  and  form  external 


FIG.  69. — Section  of  liver  from  a  case  of  actinomycosis  in  man  (CrookshanU). 


sinuses.  Or  it  may  penetrate  the  diaphragm  and  invade 
the  abdominal  organs,  causing  an  interesting  and  charac- 
teristic lesion  in  the  liver  and  other  large  viscera  (see 
Fig.  69). 

Microscopically  the  lesion  consists  chiefly  of  a  round- 


ACTINOM  YCOSIS.  265 

cell  infiltration  with  circumscribing  granulation-tissue 
leading  to  the  formation  of  cicatricial  bands.  In  the 
form  known  as  " wooden  tongue"  the  disease  runs  an 
essentially  chronic  course,  with  the  production  of  consid- 
erable amounts  of  connective  tissue. 

But  few  cases  recover,  the  disease  terminating  by  death 
from  exhaustion  or  from  complicating  pneumonia  or 
other  organic  lesions. 


CHAPTER  VI. 
MYCETOMA,  OR  MADURA-FOOT. 

A  CURIOUS  disease  of  not  infrequent  occurrence  in  the 
Indian  province  of  Scinde  is  one  known  as  mycetoma, 
Madura-foot,  or  pied  de  Madtira.  It  almost  invariably 
affects  natives  of  the  agriculturist  class,  and  in  most 
cases  begins  in  or  is  referred  by  the  patient  to  the  prick 
of  a  thorn.  It  generally  affects  the  foot,  more  rarely 
the  hand,  and  in  one  instance  was  seen  by  Boyce  in  the 
shoulder  and  hip.  It  is  more  common  in  men  than  in 
women,  individuals  between  twenty  and  forty  years  of 
age  suffering  most  frequently,  but  persons  of  any  age  or 
sex  may  suffer  from  the  disease.  It  is  insidious  in  its 
onset,  as  has  been  said,  generally  following  a  slight 
injury,  such  as  the  prick  of  a  thorn.  No  symptoms  are 
observed  in  what  might  be  called  an  incubation  stage  of 
a  couple  of  weeks'  duration,  but  after  this  time  elapses  a 
nodular  growth  gradually  forms,  attaining  in  the  course 
of  time  the  size  of  a  marble.  Its  deep  attachments  are 
indistinct  and  diffuse.  The  skin  becomes  purplish, 
thickened,  indurated,  and  adherent.  The  points  most 
frequently  invaded  at  the  onset  are  the  ball  of  the  great 
toe  and  the  pads  under  the  bases  of  the  fingers  and  toes. 

In  the  course  of  months,  although  progressing  slowly, 
the  lesions  attain  very  perceptible  size,  distinct  tumors 
being  present.  Later,  sometimes  not  until  after  a  year 
or  two,  the  nodes  begin  to  soften,  break  down,  discharge 
their  purulent  contents,  and  originate  ulcers  and  com- 
municating sinuses.  The  discharge  at  this  stage  is  a 
thin  sero-pus,  and  is  always  mixed  with  a  number  of 
fine  round  black  or  pink  bodies,  described,  when  black, 
as  resembling  gunpowder ;  when  pink,  as  resembling 

266 


MYCETOMA,    OR  MADURA-FOOT.  267 

fish-roe.  It  is  the  detection  of  these  particles  upon 
which  the  diagnosis  rests,  and  upon  which  the  divis- 
ion of  the  disease  into  the  melanoid  and  pale  varieties 
depends. 

The  progress  of  the  disease  causes  an  enormous  size 
and  a  peculiar  deformity  of  the  affected  foot  or  hand. 
The  malady  is  generally  painless. 

The  micro-organismal  nature  of  the  disease  was  early 
suspected.  In  spite  of  the  confusion  caused  by  some 
who  confounded  the  disease  with  and  described  it  as 
u  Guinea-worm, n  Carter  held  that  it  was  due  to  some 
indigenous  fungus  as  early  as  1874.  Boyce  and  Surveyor 
believe  that  the  black  particles  of  the  melanoid  variety 
represent  a  curious  metamorphosis  of  a  large  branching 
septate  fungus,  and  that  the  white  particles  of  the  other 
variety  are  the  remains  of  a  lowly-organized  fungus  and 
of  caseous  particles. 

Kanthack  tried  to  prove  the  identity  of  the  fungus 
with  the  well-known  actinomyces,  but  there  seems  to 
be  considerable  doubt  about  the  correctness  of  his  view. 

Vincent  succeeded  in  isolating  the  micro-organism 
by  puncturing  one  of  the  nodes  with  a  sterile  pipette, 
and  has  cultivated  it  upon  artificial  media.  Acid  vege- 
table infusions  seem  suitable  to  its  growth.  It  develops 
scantily  in  bouillon  at  the  room-temperature,  better  at 
37°  C. — in  from  four  to  five  days.  In  twenty  to  thirty 
days  the  colony  attains  the  size  of  a  little  pea. 

In  the  liquid  media  the  colonies  which  cling  to  the 
glass,  and  thus  remain  near  the  surface  of  the  medium, 
develop  a  rose-  or  bright-red  color. 

Cultures  in  gelatin  are  not  very  abundant,  are  colorless, 
and  are  unaccompanied  by  liquefaction. 

Upon  the  surface  of  agar-agar  strikingly  beautiful 
rounded,  glazed  colonies  are  formed.  They  are  at  first 
colorless,  but  later  become  rose-colored  or  bright  red.  The 
majority  of  the  clusters  remain  isolated,  some  of  them 
attaining  the  size  of  a  small  pea.  They  are  generally 
umbilicated  like  a  variola  pustule,  and  present  a  curious 


268 


PATHOGENIC  BACTERIA. 


appearance  when  the  central  part  is  pale  and  the  periphery 
red.  As  the  colony  ages  the  red  color  is  lost  and  it  be- 
comes dull  white.  The  colonies  are  very  adherent  to  the 
surface  of  the  medium,  and  are  said  to  be  of  cartilaginous 
consistence.  The  organism  also  grows  in  milk  without 
coagulation. 

Upon  potato  the  development  is  meagre,  slow,  and 
with  very  little  tendency  to  chromogenesis.  The  color- 
production  is  more  marked  if  the  potato  be  acid  in  reac- 


FIG.  70- — Streptothrix  Madurae  in  a  section  of  diseased  tissue  (Vincent). 

tion.  Some  of  the  colonies  upon  agar-agar  and  potato 
have  a  powdery  surface,  no  doubt  from  the  occurrence  of 
spores.  It  is,  of  course,  an  aerobic  organism.  v 

Under  the  microscope  the  organism  is  found  by  Vin- 
cent to  be  a  streptothrix — a  true  branched  fungus  con- 
sisting of  long  bacillary  branching  threads  in  a  tangled 
mass.  In  many  of  the  threads  spores  could  be  made  out. 


MYCETOMA,    OR  MADURA-FOOT.  269 

Vincent  was  unable  to  communicate  the  disease  to  animals 
by  inoculation. 

Microscopic  study  of  the  diseased  tissues  in  cases  of 
mycetoma  is  not  without  interest.  The  healthy  tissue 
is  said  to  be  sharply  separated  from  the  diseased  masses. 
The  latter  appear  as  large  degenerated  tubercles,  except 
that  they  are  extremely  vascular.  The  mycelial  or 
filamentous  fungous  mass  occupies  the  centre  of  the 
degeneration,  where  its  long  filaments  can  be  beautifully 
demonstrated  by  the  use  of  appropriate  stains,  Gram's 
method  being  excellent  for  the  purpose.  The  tissue  sur- 
rounding the  disease-nodes  is  infiltrated  with  small  round 
cells.  The  youngest  nodules  are  seen  to  consist  of  granu- 
lation-tissue, which  in  its  organization  is  checked  by  the 
coagulation-necrosis  which  is  sure  to  overtake  it.  Giant- 
cells  are  few. 

Not  infrequently  small  hemorrhages  occur  from  the 
ulcers  and  sinuses  of  the  diseased  tissues  ;  the  hemor- 
rhages can  be  explained  from  the  abundance  of  small 
blood-vessels  in  the  diseased  tissue. 

Although  the  disease  has  been  described  as  occurring 
in  Scinde,  it  is  not  limited  to  that  province,  having  been 
met  with  in  Madura,  Hissar,  Bicanir,  Dehli,  Bombay, 
Baratpur,  Morocco,  Algeria,  one  case  by  Bastini  and 
Campana  in  Italy,  and  one  by  Kempner  in  America. 


CHAPTER    VII. 
FARCIN  DU  BCEUF. 

THE  peculiar  disease  which  sometimes  affects  numbers 
of  cattle  in  Guadeloupe,  and  which  was  described  by 
the  older  writers  as  farcin  dn  bceuf,  has  been  carefully 
studied  by  Nocard.  It  is  a  disease  of  cattle  character- 
ized by  a  superficial  lymphangitis  and  lymphadenitis, 
affecting  the  tracheal,  axillary,  prescapular,  and  other 
glands.  The  affected  glands  enlarge,  suppurate,  and 
discharge  a  creamy,  sometimes  a  grumous,  pus.  The 
internal  organs  are  often  affected  with  a  pseudo-tubercu- 
losis whose  central  areas  undergo  a  purulent  or  caseous 
degeneration. 

In  the  researches  of  Nocard  it  was  discovered,  by 
staining  by  Gram's  and  by  Kuhne's  methods,  that  in 
the  centres  of  the  tubercles  micro-organisms  could  be 
defined.  They  resembled  long  delicate  filaments  rather 
intricately  woven,  characterized  by  distinct  ramifications 
which  made  clear  the  proper  classification  of  the  organ- 
ism as  a  streptothrix.  The  organism  was  successfully 
cultivated  by  Nocard  upon  various  culture-media  at  the 
temperature  of  the  body.  It  is  aerobic. 

In  bouillon  the  organism  develops  in  the  form  of  color- 
less masses  irregular  in  size  and  shape,  some  of  which 
float  upon  the  surface,  others  of  which  sink  to  the  bottom 
of  the  liquid.  Sometimes  the  surface  is  covered  by  an 
irregular  fenestrated  pellicle  of  a  gray  color. 

Upon  agar-agar  the  growth  develops  in  small,  rather 
discrete,  irregularly  rounded,  opaque  masses  of  a  yellow- 
ish-white color.  The  surfaces  of  the  colonies  are  tuber- 
culated,  and  an  appearance  somewhat  like  a  lichen  is 
observed  (see  Fig.  71). 

270 


FARCIN  DU  BCEUF.  271 

Upon  potato  very  dry  scales  of  a  pale-yellow  color 
rapidly  develop. 

The  growth  upon  blood-serum  is  less  luxuriant,  but 
similar  to  that  upon  agar-agar. 

In  milk  the  organism  produces  no  coagulation  by  its 
growth,  and  does  not  alter  the  reaction. 

Microscopic  study  always  reveals  the  organism  as  the 
same  tangled  mass  of  filaments  seen  in  the  tissues.  The 
old  cultures  are  rich  in  spores,  which  are  very  small  and 


FIG.  71. — Streptothrix  of  farcin  du  boeuf  growing  on  glycerin  agar-agar. 

develop  upon  the  most  superficial  portions  of  the  growth. 
These  spores  resist  the  penetration  of  stains  to  a  rather 
unusual  extent. 

Cultures  retain  their  virulence  for  a  long  time  :  Nocard 
found  one  virulent  after  it  had  been  kept  for  four  months 
in  an  incubating  oven  at  40°  C. 

The  Streptothrix  of  farcin  du  bceuf  is  pathogenic  for 
guinea-pigs,  cattle,  and  sheep ;  dogs,  rabbits,  horses,  and 
asses  are  immune. 

When  the  culture  or  some  pus  containing  the  micro- 


272  PATHOGENIC  BACTERIA. 

organism  is  injected  subcutaneously  into  a  guinea-pig,  a 
voluminous  abscess  results.  Not  long  afterward  the  lym- 
phatic vessels  and  glands  of  the  region  are  the  seat  of  swell- 
ing and  induration,  and  extensive  phlegmons  form,  which 
rupture  externally  and  discharge  considerable  pus.  The 
animal,  of  course,  becomes  extremely  ill  and  seems  about 
to  die ;  instead,  it  slowly  recovers  its  normal  condition. 

In  other  animals,  as  the  cow  and  the  sheep,  the  subcu- 
taneous inoculation  results  in  an  abscess  relatively  less 
extensive.  This  ulcerates,  then  indurates,  and  seems  to 
disappear,  but  after  the  lapse  of  several  weeks  or  months 
opens  again  in  the  form  of  a  new  abscess. 

In  animals  which  are  immune  or  nearly  immune,  like 
the  horse,  the  ass,  the  dog,  and  the  rabbit,  the  subcuta- 
neous inoculation  is  followed  by  the  formation  of  a  small 
abscess  which  speedily  cicatrizes. 

Intraperitoneal  inoculation  in  the  guinea-pig  gives  rise 
to  an  appearance  resembling  tuberculosis.  The  omentum 
may  be  extensively  involved  and  full  of  softened  nodes. 
The  liver,  spleen,  and  kidneys  appear  full  of  tubercles, 
but  careful  examination  will  satisfy  the  observer  that 
the  tubercles  are  only  upon  the  peritoneal  surfaces,  not 
in  the  organs. 

Intravenous  introduction  of  the  cultures  produces  a 
condition  much  resembling  general  miliary  tuberculosis. 
All  the  organs  contain  the  pseudo-tubercles  in  consider- 
able numbers. 


CHAPTER  VIII. 
RHINOSCLEROMA. 

IN  Austria,  Hungary,  Italy,  and  some  parts  of  Ger- 
many there  sometimes  occurs  a  peculiar  disease  of  the 
anterior  nares,  characterized  by  the  occurrence  of  circum- 
scribed tumors,  known  as  rhinoscleroma.  The  tumor- 
masses  are  somewhat  flattened,  isolated  or  coalescent, 
grow  with  great  slowness,  and  recur  if  excised.  The  dis- 
ease commences  in  the  mucous  membrane  and  the  adjoin- 
ing skin,  and  spreads  to  the  skin  in  the  neighborhood  by 
a  slow  invasion,  involving  the  upper  lip,  jaw,  hard  palate, 
and  sometimes  the  pharynx.  The  growths  are  without 
evidences  of  inflammation,  do  not  ulcerate,  and  consist 
microscopically  of  infiltration  of  the  papilla  and  corium 
of  the  skin,  with  round  cells  which  change  in  part  to 
fibrillar  tissue.  The  tumors  possess  a  well-developed 
lymph-vascular  system.  Sometimes  the  cells  undergo 
hyaline  degeneration. 

In  these  little  tumors  the  researches  of  Von  Frisch  dis- 
covered little  bacilli  much  resembling  both  in  morphol- 
ogy and  vegetation  the  pneumo-bacilli  of  Friedlander, 
and,  like  them,  surrounded  by  capsules.  The  only 
marked  difference  between  the  so-called  bacillus  of  rhi- 
noscleroma and  the  Bacillus  pneumonise  of  Friedlander 
is  that  the  former  stains  well  by  Gram's  method,  while 
the  latter  does  not,  and  that  the  former  is  rather  more 
distinctly  rod-shaped  than  the  latter,  and  more  often 
shows  its  capsule  in  culture-media. 

The  bacillus  can  easily  be  cultivated,  and  in  all  media 
resembles  the  bacillus  of  Friedlander  too  closely  to  be 
distinguished  from  it.  Even  when  inoculated  into  animals 
the  bacillus  behaves  much  like  Friedlander' s  bacillus. 

Inoculation  has,  so  far,  failed  to  produce  the  disease 
either  in  men  or  in  the  .lower  animals. 

18  273 


B.    THE  TOXIC    DISEASES. 


CHAPTER   I. 
TETANUS. 

ONE  of  the  most  exquisitely  toxic  bacteria  of  which 
we  have  any  knowledge  is  the  bacillus  discovered  in 
1884  by  Nicolaier,  oblained  in  pure  culture  by  Kitasato 
in  1889,  and  now  universally  recognized  as  the  cause  of 
tetanus.  It  is  a  peculiar  organism,  whose  striking  feature 
is  a  considerable  enlargement  of  one  end,  in  which  a 
bright  round  spore  is  seen  (Fig.  72).  The  bacilli  which 


FlG.  72. — Bacillus  tetani;    x  looo  (Frankel  and  Pfeiffer). 

are  not  sporiferous,  are  long,  rather  slender,  have  rounded 
ends,  seldom  unite  in  chains  or  pairs,  are  motile,  and 
have  no  flagella.  The  bacilli  stain  readily  with  ordi- 
nary aqueous  solution  of  the  anilin  dyes,  and  also  very 
readily  by  Gram's  method. 

The  tetanus  bacillus  is  a  common  saprophytic  organ- 
ism which  can  be  found  in  most  garden-earth,  in  dust, 

274 


TETANUS. 


275 


in  manure,  and  sometimes  in  the  intestinal  discharges 
of  animals.  It  is  extremely  difficult  to  isolate  and  culti- 
vate, because  it  will  not  grow  where  the  smallest  amount 
of  oxygen  is  present. 

The  method  now  generally  employed  for  the  isolation 
of  this  bacillus  is  that  originated  by  Kitasato,  and  based 
upon  his  observation  that  its  spores  can  resist  high  temper- 


FiG.  73. — Bacillus  tetani :  six-days-  FIG.  74. — Bacillus  tetani :  culture 
old  puncture-culture  in  glucose-gelatin  four  days  old  in  glucose-gelatin  (Fran- 
(Frankel  and  Pfeiffer).  kel  and  Pfeiffer). 

atures.  After  finding  that  the  typical  bacilli  are  present 
in  earth  or  pus,  or  whatever  the  material  to  be  investi- 
gated was,  Kitasato  exposed  a  portion  of  it  for  an  hour 
to  a  temperature  of  80°  C.  By  this  heating  all  the  fully- 
developed  bacteria,  tetanus  as  well  as  the  others,  and  the 


276 


PATHOGENIC  BACTERIA. 


great  majority  of  the  spores  except  those  of  tetanus,  were 
destroyed,  aud,  as  little  other  than  tetanus  spores  re- 
mained, their  cultivation  was  made  comparatively  easy. 
The  resistance  which  the  tetanus  bacilli  manifest  toward 
heat  is  only  part  of  a  great  general  resisting  power  of 
which  they  are  possessed.  It  is  said  that  they  can  retain 
their  vitality  in  the  dried  condition  for  months.  Stern- 
berg  says  they  can  resist  5  per  cent,  carbolic  solutions 
for  ten  hours,  but  will  not  grow  after  fifteen  hours'  im- 
mersion. 5  per  cent,  carbolic  acid,  to  which  0.5  per  cent. 


FIG.  75. — Bacillus  tetani :  five-days-old  colony  upon  gelatin  containing  glucose ; 
x  IOOO  (FrS.nkel  and  Pfeiffer). 

of  hydrochloric  acid  has  been  added,  destroys  them  in 
two  hours.  They  are  also  destroyed  in  three  hours  by 
i :  looo  bichlorid-of-mercury  solution  ;  but  when  to  such 
a  solution  0.5  per  cent,  of  hydrochloric  acid  is  added,  its 
activity  is  so  increased  that  the  spores  are  destroyed  in 
thirty  minutes.  The  resistance  to  heat  is  only  within 
certain  limits,  for  exposure  to  passing  steam  for  from 
five  to  eight  minutes  is  certain  to  kill  the  spores. 
The  colonies  of  the  tetanus  bacillus,  when  grown  in 


TETANUS.  277 

an  atmosphere  of  hydrogen  upon  gelatin  plates,  somewhat 
resemble  those  of  the  well-known  hay  bacillus.  There 
is  a  dense  rather  opaque  central  mass  from  which  a  more 
transparent  zone  is  readily  separable.  The  margins  of 
this  outer  zone  are  made  up  of  a  radiating  fringe  of  pro- 
jecting bacilli  (Fig.  75).  The  liquefaction  that  occurs  is 
much  slower  than  that  caused  by  bacillus  subtilis. 

When  grown  in  gelatin  puncture-cultures  the  develop- 
ment occurs  deep  in  the  puncture,  and  consists  of  mul- 
titudes of  short-pointed  processes  radiating  from  the 
puncture,  somewhat  resembling  a  fir  tree  (Fig.  73). 
Liquefaction  begins  in  the  second  week  and  causes  the 
disappearance  of  the  radiating  processes.  The  liquefac- 
tion spreads  slowly,  but  may  involve  the  entire  mass  of 
gelatin  and  resolve  it  into  a  grayish- white  syrupy  liquid, 
at  the  bottom  of  which  the  bacilli  accumulate.  The 
growth  in  gelatin  containing  glucose  is  much  more  rapid  ; 
that  in  agar-agar  punctures  is  much  slower,  but  similar 
to  the  gelatin  cultures  except  for  the  absence  of  liquefac- 
tion. The  organism  can  also  be  grown  in  bouillon,  and 
attains  its  maximum  development  at  a  temperature  of 
37°  C.  Much  gas  is  given  off  from  the  cultures. 

Cultures  of  the  tetanus  bacillus  in  all  media  give  off 
a  peculiar  characteristic  odor — a  burnt-onion  smell,  with 
a  suggestion  of  putrefaction  about  it. 

The  methods  for  excluding  the  oxygen  from  the  cul- 
tures and  replacing  it  by  hydrogen,  as  well  as  other 
methods  suggested  for  the  cultivation  of  the  strictly 
anaerobic  organisms,  are  given  under  the  appropriate 
heading  (Anaerobic  Cultures),  and  need  not  be  repeated 
here. 

A  very  simple  method  of  cultivating  the  bacillus  in 
bouillon  for  the  purpose  of  securing  a  large  amount  of 
toxin  has  been  suggested  by  the  author.1  An  ordinary 
bottle  is  filled  with  bouillon  to  the  mouth,  and  closed 
by  a  perforated  rubber  stopper  containing  a  glass  tube 

1  Centralbl.  f.  Bakt.  u.  Parasitenk.,  xix.,  Nos.  14  and  15,  April  25,  1896,  p. 
550- 


278 


PATHOGENIC  BACTERIA. 


FIG.  76.— Tetanus  bottle. . 


a   couple   of  inches   long.      Connected   with   this   glass 
tube,  by  means  of  a  short  piece  of  rubber  tubing,    is 

the  bulb  of  a  broken  pipette, 
the  other  end  of  which  is 
plugged  with  cotton  (Fig.  76). 
When  the  steam  sterilization 
takes  place  the  expanding  fluid 
ascends  -to  the  reservoir  repre- 
sented by  the  pipette  bulb,  de- 
scending again  as  the  fluid  cools. 
When  the  sterilization  is  com- 
pleted the  reservoir  is  detached, 
the  inoculation  made  by  passing 
a  very  fine  pipette  into  the  bottle, 
the  projecting  glass  tube  drawn 
out  to  a  fine  tube,  and  the  bottle 
stood  in  hot  water  until  the  ex- 
panding fluid  ascends  to  the  apex 
of  the  pointed  glass  tube.  The 
tube  is  now  sealed  in  a  flame  and  the  bottle  and  its  con- 
tents allowed  to  cool.  In  cooling  the  retracting  fluid 
leaves  a  vacuum  which  at  once  draws  up  any  minute 
bubbles  of  air  remaining,  and  allows  the  tetanus  bacillus 
to  grow  in  a  condition  of  very  fair  anaerobiosis. 

Tetanus  bacilli  exist  in  nature  as  widely  distributed 
saprophytes.  They  are  quite  common  in  the  soil,  and 
the  fact  that  they  are  most  plentiful  in  manured  ground 
has  suggested  that  they  originate  in  the  intestines  of 
horses  and  reach  the  earth  from  their  excrement.  Le 
Dentu  has,  however,  shown  that  the  tetanus  bacillus  is 
a  common  organism  in  New  Hebrides,  where  there  are  no 
horses.  In  these  islands  the  natives  poison  their  arrows 
by  dipping  them  into  a  clay  rich  in  tetanus  bacteria. 

The  work  of  Kitasato  has  given  us  a  very  exact 
knowledge  of  the  tetanus  bacillus  and  completely  estab- 
lislics  its  specific  nature. 

The  organisms  generally  enter  the  animal  body  through 
a  wound  caused  by  some  implement  which  has  been  in 


TETANUS.  279 

contact  with  the  soil,  or  enter  abrasions  from  the  soil 
directly.  Doubtless  many  of  the  wounds  are  so  small 
that  their  existence  is  overlooked,  and  this,  together 
with  the  fact  that  the  period  of  incubation  of  the  dis- 
ease, especially  in  man,  is  of  considerable  duration,  and 
at  times  permits  the  wound  to  heal  before  any  symptoms 
of  intoxication  occur,  serves  to  explain  to  us  at  least  some 
of  the  reported  cases  in  which  no  wound  is  said  to  have 
existed. 

It  would  seem  that  in  some  rare  cases  tetanus  can  occur 
without  the  previous  existence  of  a  wound.  Such  a  case 
has  been  reported  by  Kamen,  who  found  that  the  intes- 
tine of  a  person  dead  of  the  disease  was  rich  in  the 
Bacillus  tetani.  Kamen  is  of  the  opinion  that  the 
bacilli  can  grow  in  the  intestine  and  be  absorbed,  espe- 
cially where  there  are  imperfections  in  the  mucosa.  It 
is  not  impossible,  though  he  does  not  think  it  probable, 
that  the  bacteria  growing  in  the  intestine  could  elaborate 
enough  toxin  to  produce  the  disease  by  absorption. 

All  animals  are  not  alike  susceptible  to  the  disease. 
Men,  horses,  mice,  rabbits,  and  guinea-pigs  are  all  sus- 
ceptible ;  dogs  are  much  less  so.  Most  birds  are  scarcely 
at  all  susceptible  either  to  the  bacilli  or  to  the  poison. 
Amphibians  are  immune,  though  it  is  said  that  frogs 
can  be  made  susceptible  by  elevation  of  their  body- 
temperature. 

When  a  white  mouse  is  inoculated  with  an  almost 
infinitesimal  amount  of  bouillon  or  solid  culture,  or  is 
inoculated  with  garden-earth  containing  the  tetanus 
bacillus,  the  disease  is  almost  certain  to  follow,  the 
first  symptoms  coming  on  in  from  one  to  two  days. 
The  mouse  develops  typical  tetanic  convulsions,  which 
begin  first  in  the  neighborhood  of  the  inoculation,  but 
soon  become  general.  Death  follows  sometimes  in  a 
very  few  hours.  In  rabbits  the  period  of  incubation  is 
nearly  two  weeks,  and  in  man  may  be  three  weeks. 

The  conditions  in  the  animal  body  are  not  favorable 
for  the  development  of  the  bacilli,  because  of  the  free 


28o  PATHOGENIC  BACTERIA. 

supply  of  oxygen  contained  in  the  blood,  and  we  find 
that  they  grow  with  great  slowness,  remain  localized  at 
the  seat  of  inoculation,  and  never  enter  the  blood-  or 
lymph-circulation.  Doubtless  most  cases  of  tetanus  are 
cases  of  mixed  infection  in  which  the  bacillus  enters  with 
bacteria,  which  greatly  aid  its  growth  by  using  up  the 
oxygen  in  their  neighborhood.  The  amount  of  poison 
produced  must  be  exceedingly  small  and  its  power  tre- 
mendous, else  so  few  bacilli  growing  under  adverse  con- 
ditions could  not  produce  fatal  toxemia.  The  poison  is 
produced  rapidly,  for  Kitasato  found  that  if  mice  were 
inoculated  at  the  root  of  the  tail,  and  afterward  the  skin 
and  the  subcutaneous  tissues  around  the  inoculation  were 
either  excised  or  burned  out,  this  treatment  would  not 
save  the  animal  unless  the  operation  were  performed 
within  an  hour  after  the  inoculation. 

Some  incline  to  the  view  that  the  toxin  is  a  ferment, 
and  the  experiments  of  Nocard,  quoted  before  the  Acad- 
£mie  de  Me*decine,  October  22,  1895,  might  be  adduced 
in  support  of. the  theory.  He  says:  "Take  three  sheep 
with  normal  tails,  and  insert  under  the  skin  at  the  end 
of  each  tail  a  splinter  of  wood  covered  with  the  dried 
spores  of  the  tetanus  bacillus;  watch  these  animals  care- 
fully for  the  first  symptoms  of  tetanus,  then  amputate  the 
tails  of  two  of  them  20  cm.  above  the  point  of  inocula- 
tion, .  .  .  the  three  animals  succumb  to  the  disease  with- 
out showing  any  sensible  difference.'7 

The  circulating  blood  of  diseased  animals  is  fatal  to 
susceptible  animals  because  of  the  toxin  which  it  con- 
tains; and  the  fact  that  the  urine  is  also  toxic  to  mice 
proves  that  the  toxin  is  excreted  by  the  kidneys. 

From  pure  cultures  of  tetanus  bacilli  grown  in  various 
media,  and  from  the  blood  and  tissues  of  animals  affected 
with  the  disease,  Brieger  succeeded  in  separating  two 
alkaloidal  substances — "tetanin"  and  utetano- toxin, " 
both  very  poisonous  and  productive  of  tonic  convulsions; 
and  Brieger  and  Frankel  later  isolated  an  extremely  poi- 
sonous toxalbumin. 


TETANUS.  281 

The  pathology  of  the  disease  is  of  much  interest  be- 
cause of  its  purely  toxic  nature.  There  is  generally  a 
small  wound  with  a  slight  amount  of  suppuration.  At 
the  autopsy  the  organs  of  the  body  are  normal  in  appear- 
ance, except  the  nervous  system,  which  bears  the  great- 
est insult.  It,  however,  shows  little  else  than  congestion 
either  macroscopically  or  microscopically. 

An  interesting  fact  contributed  to  our  knowledge  of 
the  disease  has  been  presented  by  Vaillard  and  Rouget, 
who  found  that  if  the  tetanus  bacilli  were  introduced 
into  the  body  freed  from  their  poison,  they  were  unable 
to  produce  any  signs  of  disease  because  of  the  prompt- 
ness with  which  the  phagocytes  took  them  up.  If,  how- 
ever, their  poison  was  not  removed,  or  if  the  body-cells 
were  injured  by  the  simultaneous  introduction  of  lactic 
acid  or  other  chemical  agents,  the  bacilli  would  imme- 
diately begin  to  manufacture  the  toxin  and  produce 
symptoms  of  the  disease. 

The  toxin  is  easily  prepared,  being  readily  soluble  in 
water.  The  most  ready  method  of  preparation  is  to 
grow  the  bacilli  in  bouillon,  keeping  the  culture-medium 
at  a  temperature  of  37°  C.,  and  allowing  it  to  remain  un- 
disturbed for  from  two.  .to  four  weeks,  by  which  time  it 
will  have  attained  a  toxicity  so  great  that  0.000005  c.cm. 
will  cause  the  death  of  a  mouse.  The  toxin  is  very  rapidly 
destroyed  by  heat,  and  cannot  bear  any  temperature  above 
6o°-65°  C.  It  is  also  decomposed  by  light.  The  best 
method  of  keeping  it  is  to  add  0.5  per  cent,  of  phenol, 
and  then  store  it  in  a  cool,  dark  place.  It  will  not 
keep  its  strength  very  long  under  the  best  conditions. 

The  purified  toxin  of  Brieger  and  Cohn  was  surely 
fatal  to  mice  in  doses  of  0.00000005  gram.  Lambert,1  in 
his  comprehensive  review  of  the  use  of  tetanus  antitoxin, 
points  out  that  this  is  the  most  poisonous  substance  that 
has  ever  been  discovered. 

•  By  the  gradual  introduction  of  such  a  toxin  into  ani- 
mals Behring  and  Kitasato  have  been  able  to  produce  in 

1  New  York  Med.  Jour.,  June  5,  1897. 


282  PATHOGENIC  BACTERIA. 

their  blood  a  distinctly  potent  and  valuable  antitoxic 
substance. 

The  method  for  the  production  of  this  tetanus  anti- 
toxic serum  is  very  much  like  that  for  the  diphtheria 
antitoxic  serum  (q.  z>.),  except  that  a  much  longer  time 
is  required  for  its  production,  that  the  doses  of  toxin  are 
of  necessity  smaller  because  its  toxicity  is  greater,  and 
that  trichlorid  of  iodin  or  Gram's  solution  will  probably 
need  to  be  added  to  the  toxin  to  prevent  too  powerful  a 
local  reaction.  Horses,  dogs,  and  goats  may  be  used. 

As  tetanus  cases  are  not  very  common,  and  the  anti- 
toxic serum  when  produced  is  not  very  stable  in  its  prop- 
erties, Tizzoni  and  Cattani  have  successfully  prepared  it 
in  a  solid  form,  in  which,  it  is  claimed,  it  can  be  kept 
indefinitely,  shipped  any  distance,  and  used  after  simple 
solution  in  water.  Their  method  is  to  precipitate  the 
antitoxin  from  the  blood  of  immunized  dogs  with  alcohol. 
Numerous  cases  of  the  beneficial  action  of  this  antitoxin 
are  on  record. 

The  strength  of  the  serum  is  generally  expressed 
i  :  1,000,000,  i  :  10,000,000,  etc.,  which  indicates  that  i 
c.cm.  of  the  serum  is  capable  of  protecting  1,000,000  or 
10,000,000  grams  of  guinea-pig  from  infection. 

The  experiments  of  Alexander  Lambert  show  that  a 
protective  power  of  i  :  800,000,000  can  be  attained. 

As  Welch  has  pointed  out,  the  antitoxin  of  tetanus  has 
proved  to  be  rather  a  disappointment  in  human  medicine, 
and  also  for  the  treatment  of  large  animals,  such  as  the 
horse.  The  results  following  its  injection,  in  combination 
with  the  sterile  toxin,  into  mice,  guinea-pigs,  and  rabbits 
are  highly  satisfactory,  but  the  amount  needed,  in  pro- 
portion to  the  body-weight,  to  save  the  animal  from  the 
toxin  being  manufactured  in  its  body  by  bacilli  increases 
so  enormously  with  the  day  or  hour  of  the  disease  as  to 
make  the  dosage,  which  increases  millions  of  times  where 
that  of  diphtheria  antitoxin  increases  but  tenfold,  a  matter 
of  difficulty  and  uncertainty.  Nocard  also  calls  atten- 
tion to  the  fact  that  the  existence  of  tetanus  is  unknown 


TETANUS.  283 

until  there  is  sufficient  toxemia  to  produce  spasms,  and 
that  therefore  it  is  impossible  to  attack  the  disease  in  its 
inception ;  we  are  obliged  to  meet  it  upon  the  same 
grounds  as  diphtheria  in  the  later  days  of  the  disease — 
a  time  when  it  is  well  known  that  the  chances  of  im- 
provement are  greatly  lessened. 

Of  course,  as  there  is  no  other  remedy  that  combats 
the  disease  at  all,  the  antitoxin  is  one  which,  when  ob- 
tainable, should  always  be  employed. 

An  interesting  observation  has  been  recently  made  by 
Wasserman,1  who,  assuming  that  the  destruction  of 
nerve-cells  in  the  cerebrum  and  cord  during  tetanus  tox- 
emia might  have  something  to  do  with  immunity,  be- 
lieved it  possible  to  obtain  from  these  cells  an  immuniz- 
ing substance.  Investigating  the  subject,  he  found  that 
when  fresh  brain  or  spinal  cord  was  rubbed  up  in  a  mor- 
tar with  physiological  salt  solution,  and  injected  into  ani- 
mals, the  mixture  had  the  power  not  only  to  confer  upon 
them  an  immunity  lasting  for  twenty-four  hours,  but  also 
was  potent  enough  to  neutralize  the  effects  of  an  injec- 
tion of  tetanus  toxin  ten  times  as  large  as  that  necessary 
to  kill  the  animal  in  doses  of  i  c.cm. 

These  observations  may  offer  a  possible  solution  of  the 
difficult  problem  laid  before  us  by  Montesano  and  Mon- 
tesson,2  who  unexpectedly  found  the  tetanus  bacillus  in 
pure  culture  in  the  cerebro-spinal  fluid  of  a  case  of  para- 
lytic dementia  that  died  without  a  tetanic  symptom. 

1  Berlin,  klin.   Wochenschrift,  1898,  No.  I. 

2  Centralbl.f.  Bakt,,  u.  Parasitenk.,  Bd.  xxii.,  Nos.  22,  23,  p.  663.  Dec.,  1897. 


CHAPTER  II. 
DIPHTHERIA. 

IN  1883,  Klebs  pointed  out  the  existence  of  a  bacillus 
in  the  pseudo-membranes  upon  the  fauces  of  patients 
suffering  from  diphtheria,  but  it  was  not  until  1884  that 
Loffler  succeeded  in  isolating  and  cultivating  the  organ- 
ism, which  is  now  known  by  both  their  names — the 
Klebs-Loffler  bacillus. 

The  bacillus  as  described  by  Loffler  is  about  the  length 
of  the  tubercle  bacillus,  about  twice  its  diameter,  has  a 


FIG.   77.— Bacillus   diphtheriae,  from  a  culture   upon   blood-serum;    x  1000 
(Frftnkel  and  Pfeiffer). 

curve  similar  to  that  which  characterizes  the  tubercle 
bacillus,  and  has  rounded  ends  (Fig.  77).  It  does  not 
form  chains,  though  two,  three,  and  rarely  four  individ- 
uals may  be  found  joined ;  generally  the  individuals  are 
all  separate  from  one  another.  The  morphology  of  the 
bacillus  is  peculiar  in  its  considerable  irregularity,  for 


DIPHTHERIA.  285 

among  the  well-formed  individuals  which  abound  in 
fresh  cultures  a  large  number  of  peculiar  organisms  are 
to  be  found,  some  much  larger  than  normal,  some  with 
one  end  enlarged  to  a  club-shape,  some  greatly  elongated, 
with  both  ends  expanded  into  club-shaped  enlargements. 
These  bizarre  forms  seem  to  represent  an  involution-form 
of  the  organism,  for,  while  present  in  perfectly  fresh  cul- 
tures, they  are  so  abundant  in  old  cultures  that  scarcely 
a  single  well-formed  bacillus  can  be  found.  It  not  infre- 
quently happens  that  in  unstained  bacilli  distinct  gran- 
ules can  be  defined  at  the  ends — polar  granules — thus 
giving  the  organism  somewhat  the  appearance  of  a 
diplococcus. 

The  bacillus  can  be  readily  stained  by  aqueous  solu- 
tions of  the  anilin  colors,  but  more  beautifully  and 
characteristically  with  Loffler's  alkaline  methylene  blue  : 

Saturated  alcoholic  solution  of  methylene  blue,    30 ; 
i :  10,000  aqueous  solution  of  caustic  potash,     100 ; 

and  an  aqueous  solution  of  dahlia,  as  recommended  by 
Roux. 

When  cover-glass  preparations  are  stained  with  these 
solutions,  the  bizarre  forms  already  mentioned  are  much 
more  obvious  than  in  the  unstained  individuals,  and 
the  contrast  between  the  polar  granules,  which  color  in- 
tensely, and  the  remainder  of  the  bacillus,  which  tinges 
slightly,  is  marked.  Through  good  lenses  the  organisms 
are  always  distinct  bacilli,  notwithstanding  the  fact  that 
the  ends  stain  more  deeply  than  the  centres,  and  it  is 
only  through  poor  lenses  that  the  organisms  can  be  mis- 
taken for  diplococci.  The  bacilli  stain  well  by  Gram's 
method,  this  being  a  good  method  to  employ  for  their 
definition  in  sections  of  tissue,  though  Welch  and  Abbott 
assert  that  Weigert's  fibrin  method  and  picro-carmin  give 
the  most  beautiful  results. 

The  diphtheria  bacillus  does  not  form  spores,  and  is 
delicate  in  its  thermal  range.  Loffler  found  that  it  could 
not  endure  a  temperature  of  60°  C.,  and  Abbott  has  shown 


286  PATHOGENIC  BACTERIA. 

that  a  temperature  of  58°  C.  for  ten  minutes  is  fatal  to  it. 
Notwithstanding  this  susceptibility,  the  organism  can 
be  kept  alive  for  several  weeks  after  being  dried  upon 
shreds  of  silk  or  when  surrounded  by  dried  diphtheria 
membrane. 

No  flagella  have  been  demonstrated  upon  the  bacillus. 
It  is  non-motile. 

Fernbach  has  shown  that  when  the  organisms  are 
grown  in  a  medium  exposed  to  a  passing  current  of  air, 
the  luxuriance  of  their  development  is  increased,  though 
their  life-cycle  is  shorter.  The  growth  can  also  take 
place  when  the  air  is  excluded,  so  that  the  bacillus  must 
be  classed  among  the  optional  anaerobic  organisms. 

The  diphtheria  bacillus  grows  readily  upon  all  the 
ordinary  media,  and  is  a  very  easy  organism  to  obtain 
in  pure  culture.  Loffler  has  shown  that  the  addition 
of  a  small  amount  of  glucose  to  the  culture-medium 
increases  the  rapidity  of  the  growth,  and  suggests  a 
special  medium  which  bears  his  name — Loffler's  blood- 
serum  mixture : 

Blood-serum,  3 ; 

Ordinary  bouillon  -f  i  per  cent,  of  glucose,     i. 

This  mixture  is  filled  into  tubes,  coagulated,  and  steril- 
ized like  blood-serum,  and  is  one  of  the  best-known  media 
in  connection  with  the  study  of  diphtheria. 

The  studies  of  Michel l  have  shown  that  the  develop- 
ment of  the  culture  is  much  more  luxuriant  and  rapid 
when  horse  serum  instead  of  beef  or  calves'  blood  is  used. 
Horse's  blood  can  easily  be  secured  by  the  introduction  of 
a  trocar  into  the  jugular  vein  ;  5  liters  of  it  can  be  with- 
drawn without  causing  the  animal  any  inconvenience  or 
producing  symptoms. 

The  impossibility  of  clinically  making  an  accurate  di- 
agnosis of  diphtheria  without  a  bacteriologic  examination 
has  caused  main  private  physicians  and  many  medical 
societies  and  boards  of  health  to  equip  laboratories  where 

1  CtrtraM.f.  Bakt.  u.  Parasilcnk.,  Sept.  24,  1897,  Bd.  xxii.,  Nos.  loand  II. 


DIPHTHERIA.  287 

accurate  examinations  can  be  made.  The  method  re- 
quires some  apparatus,  though  a  competent  bacteriologist 
can  often  make  shift  with  a  bake-oven,  a  wash-boiler, 
and  other  household  furniture  instead  of  the  regular 
sterilizers  and  incubators,  which  are  expensive. 

When  it  is  desired  to  make  a  bacteriologic  diagnosis 
of  a  suspected  case  of  diphtheria  or  to  secure  the  bacillus 
in  pure  culture,  a  sterile  platinum  wire  having  a  small 
loop  at  the  end,  or  a  swab  made  by  wrapping  a  little 
cotton  around  the  end  of  a  piece  of  wire  and  carefully 
sterilizing  in  a  test-tube,  is  introduced  into  the  throat 
and  touched  to  the  false  membrane,  after  which  it  is 
smeared  carefully  ovei  the  surface  of  at  least  three  of 
the  blood-serum-mixture  tubes,  without  either  again 
touching  the  throat  or  being  sterilized.  The  tubes  thus 
inoculated  are  stood  away  in  an  incubating  oven  at  the 
temperature  of  37°  C.  for  twelve  hours,  then  examined. 
If  the  diphtheria  bacillus  is  present  upon  the  first  and 
second  tubes,  there  will  be  a  smeary  yellowish-white  layer, 
with  outlying  colonies  on  the  second  tube,  while  the  third 
tube  will  show  rather  large  isolated  whitish  or  slightly 
yellowish  colonies,  smooth  in  appearance,  but  rather  ir- 
regular in  outline.  Very  often  the  colonies  are  china- 
white  in  appearance.  These  colonies,  if  found  by  micro- 
scopic examination  to  be  made  up  of  diphtheria  bacilli, 
will  confirm  the  diagnosis  of  diphtheria,  and  will  at  the 
same  time  give  pure  cultures  when  transplanted.  There 
are  very  few  other  bacilli  which  grow  so  rapidly  upon 
Loffler's  mixture,  and  scarcely  one  other  which  is  found 
in  the  throat. 

Ohlmacher  recommends  the  microscopic  examination 
of  the  still  invisible  growth  in  five  hours.  A  platinum 
loop  is  nibbed  over  the  inoculated  surface ;  the  material 
secured  is  then  mixed  with  distilled  water,  dried  on  a 
cover-glass,  stained  with  methylene  blue,  and  examined. 
This  method,  if  reliable,  will  be  very  valuable  in  making 
an  early  diagnosis  preparatory  to  the  use  of  the  antitoxin. 

The  presence  of  diphtheria  bacilli  in  material  taken 


288 


PATHOGENIC  BACTERIA. 


from  the  throat  does  not  necessarily  prove  the  patient  to 
be  diseased.  Virulent  bacilli  can  often  be  discovered  in 
the  throats  of  healthy  persons  who  have  knowingly  or 
unknowingly  come  in  contact  with  the  disease.  The 
bacteriologic  examination  is  only  an  adjunct  to  the 
,  clinical  diagnosis,  and  must  never  be  taken  as  positive 
in  itself. 

The  bacillus  grows  similarly  upon  blood-serum  and 
Loffler's  mixture.  Upon  glycerin  agar-agar  and  agar-agar 
the  colonies  are  much  larger,  more  translucent,  always 


FIG.  78.— Diphtheria  bacilli  (from  photographs  taken  by  Prof.  E.  K.  Dun- 
ham, Carnegie  Laboratory,  New  York) :  a,  pseudo-bacillus ;  b,  true  bacillus ; 
f,  pseudo-bacillus. 

without  the  yellowish-white  or  china-white  color  of  the 
blood-serum  cultures,  and  generally  are  distinctly  divided 
into  a  small  elevated  centre  and  a  flatter  surrounding  zone 
with  indented  edges,  sometimes  with  a  distinctly  radiated 


DIPHTHERIA.  289 

appearance.  It  must  be  remarked  that  when  sudden 
transplantations  are  made  from  blood-serum  to  agar- 
agar  the  growth  resulting  is  meagre,  but  the  oftener 
this  growth  is  transplanted  to  fresh  agar-agar  the  more 
luxuriant  it  becomes. 

The  growth  in  gelatin  puncture-cultures  is  character- 
ized by  small  spherical  colonies  which  develop  along  the 
entire  length  of  the  needle-track.  The  gelatin  is  not 
liquefied. 

Upon  the  surface  of  gelatin  plates  the  colonies  that 
develop  do  not  attain  anything  like  the  size  of  the  colo- 
nies upon  Loffler's  mixture.  They  appear  to  the  naked 


FIG.    79. — Bacillus  diphtheriae,  colony  twenty-four  hours  old  upon  agar-agar; 
x  100  (Frankel  and  Pfeiffer). 

eye  as  whitish  points  with  smooth  contents  and  regular 
though  sometimes  indented  borders.  Under  the  micro- 
scope they  appear  as  granular,  yellowish-brown  colonies 
with  irregular  borders  (Fig.  79). 

When  planted  in  bouillon  the  organism  causes  a  diffuse 
cloudiness  at  first,  but,  not  being  motile,  soon  settles  to 
the  bottom  in  the  form  of  a  rather  flocculent  precipitate 
which  has  a  tendency  to  cling  to  the  sides  of  the  glass. 
Sometimes  a  delicate  irregular  mycoderma  .  forms  upon 

19 


290  PATHOGENIC  BACTERIA. 

the  surface,  especially  when  the  cultivation  is  made  by 
the  method  of  Fernbach  with,  a  passing  current  of  air. 
This  mycoderma,  which  may  appear  quite  regular  when 
the  flask  is  undisturbed,  is  so  brittle  that  it  at  once  falls 
to  pieces  if  the  flask  be  moved. 

Spronck  has  recently  determined  that  the  characteris- 
tics of  the  growth  of  the  diphtheria  bacillus  in  bouillon, 
as  well  as  the  amount  of  toxin-production,  vary  accord- 
ing to  the  amount  of  glucose  in  the  bouillon.  He  divides 
the  cultures  into  three  types  : 

Type  A.  The  reaction  of  the  bouillon  becomes  acid 
and  remains  acid,  the  acidity  increasing.  The  bacilli 
accumulate  at  the  bottom  of  the  clear  liquid.  The 
toxin-production  is  meagre. 

Type  B.  There  is  no  change  from  alkalinity  to  acidity, 
but  the  original  alkalinity  of  the  bouillon  steadily  in- 
creases. The  culture  is  very  rich,  the  bottom  of  the 
flask  shows  a  considerable  sediment,  the  liquid  is  cloudy, 
and  a  delicate  growth  occupies  the  surface.  The  toxicity 
is  very  great. 

Type  C.  In  a  few  days  the  reaction  of  the  culture 
becomes  acid,  and  then  later  on  changes  to  alkaline. 
During  the  acid  period  the  liquid  is  clear,  with  a  white 
surface-growth.  When  the  alkalinity  returns  the  bouillon 
clouds  and  the  surface-growth  increases  in  thickness. 
Sediment  accumulates  at  the  bottom  of  the  flask.  The 
toxicity  of  the  culture  is  much  less  than  in  Type  B. 

Spronck  regards  the  varying  reaction  as  due  to  the 
fermentation  of  the  glucose,  and  asserts  that  the  most 
luxuriant  and  toxic  cultures  are  those  in  which  no 
glucose  is  present.  To  exclude  as  much  of  the  undesir- 
able sugar  as  possible,  he  makes  the  bouillon  from  the 
stalest  meat  obtainable,  preferring  it  when  just  about  to 
putrefy.  Of  the  meats  experimented  with,  beef  was 
found  to  be  the  best. 

In  large  cities  meat  is  ordinarily  kept  sufficiently  long 
before  being  offered  for  sale  to  meet  Spronck's  require- 
ment. 


DIPHTHERIA.  291 

Upon  potato  the  bacillus  develops  only  when  the  reac- 
tion is  alkaline.  The  potato-growth  is  not  characteristic. 
Welch  and  Abbott  always  secured  a  growth  of  the  organ- 
ism when  planted  upon  potato,  but  do  not  mention  the 
reaction  of  the  medium  they  employed. 

Milk  is  an  excellent  medium  for  the  cultivation  of  the 
Bacillus  diphtheriae,  and  is  possibly  at  times  a  means  of 
infection.  Litmus  milk  is  an  excellent  medium  for  ob- 
serving the  changes  of  reaction  brought  about  by  the 
growth  of  the  bacillus.  At  first  the  alkalinity,  which 
is  always  favorable  to  the  development  of  the  bacillus, 
is  destroyed  by  the  production  of  an  acid.  When  the 
culture  is  old  the  acid  is  replaced  by  a  strong  alkaline 
reaction. 

Palmirski  and  Orlowski l  assert  that  the  bacillus  pro- 
duces indol,  but  only  after  the  third  week.  Smith,  how- 
ever, came  to  a  contrary  result,  and  found  that  when 
diphtheria  bacillus  grew  in  the  dextrose-free  bouillon 
that  he  recommends  no  indol  was  produced.2 

Diphtheria  as  it  occurs  in  man  is  generally  a  disease 
characterized  by  the  formation  of  a  pseudomembrane 
upon  the  fauces.  It  is  a  local  infection,  due  to  the 
presence  and  development  of  the  bacilli  in  the  pseudo- 
membrane,  but  is  accompanied  by  a  general  toxemia 
resulting  from  the  absorption  of  a  violently  poisonous 
substance  produced  by  the  bacilli.  The  bacilli  are  found 
only  in  the  membranous  exudation,  and  most  plentifully 
in  its  older  portions.  As  a  rule,  they  do  not  distribute 
themselves  through  the  circulation  of  the  animal,  though 
at  times  they  may  be  found  in  the  heart's  blood. 

The  most  malignant  cases  of  the  disease  are  thought 
to  be  due  to  pure  infection  by  the  diphtheria  bacillus, 
though  such  cases  are  more  rare  than  those  in  which  the 
Streptococcus  pyogenes  and  Staphylococcus  aureus  and 
albus  are  found  in  association  with  it. 

In  a  series  of  234  cases  carefully  and  statistically  studied 

1  Centralbl.f.  Bakt.  u.  Parasitenk.,  Mar.,  1895. 

2  Jour,  of  Exper.  Med.t  vol.  ii.,  No.  5,  Sept.,  1897,  p.  546. 


292  PATHOGENIC  BACTERIA. 

by  Blasi  and  Russo-Travali  it  was  found  that  in  26  cases 
of  pseudomenibranous  angina  due  to  streptococci,  staphy- 
lococci,  colon  bacilli,  and  pneumococci,  2  patients  died, 
the  mortality  being  3.84  per  cent.  In  102  cases  of  pure 
diphtheria  28  died,  a  mortality  of  27.45  per  cent.  Seventy- 
six  cases  showed  diphtheria  bacilli  and  staphylococci;  of 
these,  25,  or  32.89  per  cent,  died.  Twenty  cases  showed 
the  diphtheria  bacilli  and  Streptococcus  pyogenes,  with  6 
deaths — 30  per  cent.  In  7  cases,  of  which  3,  or  43  per 
cent.,  were  fatal,  the  diphtheria  bacillus  was  in  com- 
bination with  streptococci  and  pneumococci.  The  most 
dangerous  forms  met  were  3  cases,  all  fatal,  in  which  the 
diphtheria  bacillus  was  found  in  combination  with  the 
Bacillus  coli. 

It  may  be  well  to  remark  that  all  pseudomembranous 
diseases  of  the  throat  are  not  diphtheria,  but  that  some 
of  them,  exactly  similar  in  clinical  picture,  result  from 
the  activity  of  the  pyogenic  organisms  alone,  and  are 
neither  diphtheria  nor  contagious. 

Diphtheritic  inflammations  of  the  throat  are  not  always 
accompanied  by  the  formation  of  the  usual  pseudomem- 
brane,  it  rarely  but  occasionally  happening  that  in  the 
larynx  a  rapid  inflammatory  edema  without  a  fibrinous 
surface-coating  causes  a  fatal  suffocation.  Only  a  bac- 
teriological examination  will  reveal  the  nature  of  the 
disease  in  such  cases. 

Herman  Biggs,1  in  an  interesting  discussion  of  the 
occurrence  of  the  diphtheria  bacillus  and  its  relation  to 
diphtheria,  comes  to  the  following  conclusions: 

1.  "  When  the  diphtheria  bacillus  is  found  in  healthy 
throats  investigation  almost  always  shows  that  the  indi- 
viduals have  been  in  contact  with  cases  of  diphtheria. 
The  presence  of  the  bacillus  in  the  throat,  without  any 
lesion,  does  not,  of  course,  indicate  the  existence  of  the 
disease. 

2.  "The  simple  anginas  in  which  virulent  diphtheria 
bacilli  are  found  are  to  be  regarded  from  a  sanitary  stand- 

1  Amer.  Jour,  of  the  AffJ.  Sciences,  Oct.,  1896,  vol.  xxii.,  No.  4,  p.  41 1. 


DIPHTHERIA.  293 

point  in  exactly  the  same  way  as  the  cases  of  true  diph- 
theria. 

3.  u  Cases  of  diphtheria  present  the  ordinary  clinical  feat- 
ures of  diphtheria,  and  show  the  Klebs-Iy6ffler  bacilli. 

4.  "Cases  of  angina   associated  with  the   production 
of  membrane  in  which  no  diphtheria  bacilli  are  found 
might  be  regarded  from   a  clinical  standpoint  as  diph- 
theria, but  bacteriological  examination  shows  that  some 
other  organism   than   the   Klebs-Loffler  bacillus   is  the 
cause  of  the  process." 

No  more  convincing  proof  of  the  existence  of  a  power- 
ful poison  in  diphtheria  could  be  desired  than  the  evi- 
dences of  general  toxemia  resulting  from  the  absorption 
of  material  from  a  comparatively  small  number  of  bacilli 
situated  upon  a  little  patch  of  mucous  membrane. 

In  animals  artificially  inoculated  the  lesions  produced 
are  not  identical  with  those  seen  in  the  human  subject, 
yet  they  have  the  same  general  features  of  local  infection 
with  general  toxemia. 

Guinea-pigs,  kittens,  and  young  pups  are  susceptible 
animals.  When  half  a  cubic  centimeter  of  a  twenty-four- 
hour-old  bouillon  culture  is  injected  beneath  the  skin  of 
such  an  animal,  the  bacilli  multiply  at  the  point  of  in- 
oculation, with  the  production  of  a  patch  of  inflamma- 
tion associated  with  a  distinct  fibrinous  exudation  and 
the  presence  of  an  extensive  edema.  The  animal  dies  in 
twenty-four  to  thirty-six  hours.  The  liver  is  enlarged, 
and  sometimes  shows  minute  whitish  points,  which  in 
microscopic  sections  prove  to  be  necrotic  areas  in  which 
the  cells  are  completely  degenerated  and  the  chromatin  of 
their  nuclei  is  scattered  about  in  granular  form.  Similar 
necrotic  foci,  to  which  attention  was  first  called  by  Oertel, 
are  present  in  nearly  all  the  organs  in  cases  of  death  from 
the  toxin.  The  bacilli  are  constantly  absent  from  these 
lesions.  Welch  and  Flexner l  have  shown  these  foci  to 
be  common  to  numerous  irritant  poisonings,  and  not 
peculiar  to  diphtheria. 

1  Bull,  of  the  Johns  Hopkins  Hospital,  Aug.,  1891. 


294  PA-  THOGENIC  BA  CTERIA . 

The  lymphatic  glands  are  usually  enlarged  ;  the  adrenals 
are  also  enlarged,  and,  in  cases  into  which  the  live  bacilli 
have  been  injected,  are  hemorrhagic. 

Sometimes  the  bacilli  themselves  are  present  in  the 
internal  organs,  and  even  in  the  blood,  but  generally  this 
is  not  the  case. 

It  might  be  argued,  from  the  different  clinical  pictures 
presented  by  the  disease  as  it  occurs  in  man  and  in 
animals,  that  they  were  not  expressions  of  the  same 
thing.  A  careful  study,  however,  together  with  the  evi- 
dences adduced  by  Roux  and  Yersin,  who  found  that 
when  the  bacilli  were  introduced  into  the  trachea  of 
animals  opened  by  operation  a  typical  false  membrane 
was  produced,  and  that  diphtheritic  palsy  often  followed, 
and  of  hundreds  of  investigators,  who  find  the  bacilli 
constantly  present  in  the  disease  as  it  occurs  in  man, 
must  satisfy  us  that  the  doubt  of  the  etiological  role  of 
the  bacillus  rests  on  a  very  slight  foundation. 

All  possible  skepticism  of  the  specificity  of  the  bacillus 
on  my  part  was  dispelled  by  an  accidental  infection  that 
kept  me  housed  for  three  weeks  during  the  busiest  season 
of  the  year.  Without  having  been  exposed  to  any  known 
diphtheria  contagion,  while  experimenting  in  the  labora- 
tory, a  living  virulent  culture  of  the  diphtheria  bacillus 
was  drawn  into  a  pipette  and  accidentally  entered  my 
mouth.  Through  carelessness  no  precautions  were  taken 
to  prevent  serious  consequences,  and  as  a  result  of  the 
accident,  two  days  later,  my  throat  was  full  of  typical 
pseudomembrane  which  private  and  Health  Board  bac- 
teriological examinations  showed  contained  pure  cultures 
of  the  Klebs-Loffler  bacilli. 

One  reason  for  skepticism  in  this  particular  is  the 
supposed  existence  of  a  faeucfodiphthtria  bacillus,  which 
has  so  many  points  in  common  with  the  real  diphtheria 
bacillus  that  it  is  difficult  to  distinguish  between  them. 
We  have,  however,  come  to  regard  this  pseudobacillus  as 
an  attenuated  form  of  the  real  bacillus.  The  chief  points 
of  difference  between  the  bacilli  are  that  the  pseudo- 


DIPHTHERIA.  295 

bacillus  seems  to  be  shorter  than  the  diphtheria  bacillus 
when  grown  upon  blood-serum;  that  the  cultures  in 
bouillon  seem  to  progress  much  more  rapidly  at  a  tem- 
perature of  from  2o°-22°  C.  than  those  of  the  true  bacillus; 
and  that  the  pseudobacillus  is  not  pathogenic  for  ani- 
mals. These  slight  distinctions  are  all  exactly  what 
should  be  expected  of  an  organism  whose  virulence  had 
been  lost,  and  whose  vegetative  powers  had  been  altered, 
by  persistent  manipulation  or  by  unfavorable  surround- 
ings. 

Park l  carefully  studied  this  subject,  and  found  that 
all  bacilli  with  the  exact  morphology  of  the  diphtheria 
bacillus,  found  in  the  human  throat,  are  virulent  Klebs- 
LofHer  bacilli,  while  forms  found  in  the  throat  closely 
resembling  them,  but  more  uniform  in  size  and  shape, 
shorter  in  length,  and  of  more  equal  staining  properties 
with  Loffler's  alkaline  methylene  blue  solution,  can  be, 
with  reasonable  safety,  regarded  as  pseudodiphtheria 
bacilli,  especially  if  it  be  found  that  they  produce  an 
alkaline  rather  than  an  acid  reaction  by  their  growth  in 
bouillon.  The  pseudodiphtheria  bacilli  were  found  in 
about  i  per  cent,  of  throats  examined  in  New  York;  they 
seem  to  have  no  relationship  to  diphtheria.  They  are 
never  virulent. 

A  difference  of  possibly  much  greater  importance  is  that 
observed  by  Martini,2  that  the  diphtheria  bacillus  will  not 
grow  in  fluid  antitoxic  serum  in  which  the  pseudodiph- 
theria bacillus  thrives.  Both  the  real  and  the  pseudoba- 
cilli  flourish  upon  coagulated  antitoxic  serum.  If  this  dif- 
ference in  the  behavior  of  the  bacilli  bears  any  relation 
to  the  so-called  "specific  immunity-reaction"  of  cholera 
and  typhoid  fever,  it  may  be  of  great  future  importance. 

The  diphtheria  bacilli  are  always  present  in  the  throats 
of  patients  suffering  from  diphtheria,  and  constitute  the 
element  of  contagion  by  being  accidentally  discharged 
by  the  nose  or  mouth  by  coughing,  sneezing,  vomiting, 

1  Scientific  Bulletin  No.  i,  Health  Department,  City  of  New  York,  1895. 
a  Centralbl.f.  Bakt.  u.  Parasitenk.,  Bd.  xxi.,  No.  3,  Jan.  30,  1897. 


296  PATHOGENIC  BACTERIA. 

etc.  Whoever  comes  in  contact  with  such  materials  is 
in  danger  of  infection. 

It  is  of  great  interest  to  notice  the  remarkable  results 
obtained  by  Biggs,  Park,  and  Beebe  in  New  York,  where 
the  bacteriological  examinations  conducted  in  connection 
with  diphtheria  show  that  the  virulent  bacilli  may  be 
found  in  the  throats  of  convalescents  as  long  as  five  weeks 
after  the  discharge  of  the  membrane  and  the  commence- 
ment of  recovery,  and  that  they  exist  not  only  in  the 
throats  of  the  patients  themselves,  but  also  in  the  throats 
of  their  care-takers,  who,  while  not  themselves  infected, 
may  be  the  means  of  conveying  the  disease  from  the 
sick-room  to  the  outer  world.  Even  more  extraordinary 
are  the  observations  of  Hewlett  and  Nolen,1  who  found 
the  bacilli  in  the  throats  of  patients  seven,  nine,  and  in 
one  case  twenty-three  iceeks  after  convalescence.  The 
importance  of  this  observation  must  be  apparent  to  all 
readers,  and  serves  as  further  evidence  why  most  thor- 
ough isolation  should  be  practised  in  connection  with 
this  dreadful  disease. 

Park  2  found  virulent  diphtheria  bacilli  in  about  i  per 
cent,  of  the  healthy  throats  examined  in  New  York  City. 
Diphtheria  was,  however,  prevalent  in  the  city  at  the 
time.  Most  of  the  persons  in  whose  throats  they  existed 
had  been  in  direct  contact  with  cases  of  diphtheria.  Very 
many  of  those  whose  throats  contained  the  virulent  bacilli 
did  not  develop  diphtheria.  He  concludes  that  the  mem- 
bers of  a  household  in  which  a  case  of  diphtheria  exists 
should  be  regarded  as  sources  of  danger,  unless  cultures 
from  their  throats  show  the  absence  of  diphtheria  bacilli. 

In  connection  with  the  contagiousness  of  diphtheria 
the  recent  experiments  of  Reyes  are  interesting.  He 
has  demonstrated  that  in  absolutely  dried  air  distributed 
diphtheria  bacilli  die  in  a  few  hours.  Under  ordinary 

1  Brit.  M  :     /  MT.,  Feb.  i,  1896. 

5  Report  on   Bacteriological    Investi-jntions   and    Diagnosis   of  Diphtheria, 
I  iv  4,  1893,  to  May  4,  1894,  Scientific  Bulletin  No.  /,  Health  Depart- 
ment, City  of  New  York. 


DIPHTHERIA.  297 

conditions  their  vitality,  when  dried  on  paper,  silk,  etc., 
continues  for  a  few  days.  In  air  that  is  moist  the  dura- 
tion of  vitality  is  prolonged  to  about  a  week.  In  sand 
exposed  to  a  dry  atmosphere  they  die  in  five  days  in  the 
light;  in  sixteen  to  eighteen  days  in  the  dark.  When 
the  sand  is  exposed  to  a  moist  atmosphere  the  duration 
of  vitality  is  doubled.  In  fine  earth  they  remained  alive 
seventy-five  to  one  hundred  and  five  days  in  dry  air,  and 
one  hundred  and  twenty  days  in  moist  air. 

From  time  to  time  reference  has  been  made  to  the 
toxin  elaborated  by  the  diphtheria  bacillus.  Roux  and 
Yersin  first  demonstrated  the  existence  of  this  substance 
in  cultures  passed  through  a  Pasteur  porcelain  filter. 
The  toxin  is  intensely  poisonous;  it  is  not  an  albumin- 
ous substance,  and  can  be  elaborated  by  the  bacilli 
when  grown  in  non-albuminous  urine,  or,  as  suggested 
by  Uschinsky,  in  non-albuminous  solutions  whose  prin- 
cipal ingredient  is  asparagin.  The  toxic  value  of  the 
cultures  is  greatest  in  the  second  or  third  week. 

In  addition  to  the  toxin,  a  toxalbumin  has  been  isolated 
by  Brieger  and  Frankel. 

Bearing  discovered  that  the  blood  of  animals  rendered 
immune  to  diphtheria  by  inoculation,  first  with  attenu- 
ated and  then  with  virulent  organisms,  contained  a  neu- 
tralizing substance  which  was  capable  of  annulling  the 
effects  of  the  bacilli  or  the  toxin  when  simultaneously  or 
subsequently  inoculated  into  non-protected  animals.  This 
substance,  in  solution  in  the  blood-serum  of  the  immu- 
nized animals,  is  the  diphtheria  antitoxin. 

The  preparation  of  the  antitoxin  for  therapeutic  pur- 
poses received  a  further  elaboration  in  the  hands  of  Roux. 
The  subject  is  one  of  great  interest,  but  must  be  consid- 
ered briefly  in  a  work  of  this  kind. 

The  antitoxin  is  manufactured  commercially  at  present, 
the  method  being  the  immunization  of  large  animals  to 
great  quantities  of  the  toxin,  and  the  withdrawal  of  their 
antitoxic  blood  when  the  proper  degree  of  immunity  has 
been  attained.  The  details  are  as  follows : 


298  PATHOGENIC  BACTERIA. 

The  Preparation  of  the  Toxin.—  The  method  employed 
by  Roux  and  others  at  the  present  time  was  first  sug- 
gested by  Fernbach,  and  consists  in  growing  the  most 
virulent  bacilli  obtainable  in  alkaline  bouillon  exposed 
in  a  thin  layer  to  the  passage  of  a  current  of  air. 

The  cultures  are  allowed  to  grow  for  three  or  four 
weeks  at  a  temperature  of  37°  C.,  with  a  stream  of 
moist  air  constantly  passing  over  them.  After  the  given 
time  has  passed,  it  will  be  found  that  the  acidity  prima- 
rily produced  by  the  bacillus  gives  place  to  a  much  more 
intense  alkalinity  than  originally  existed.  The  acme  of 
the  toxin-production  seems  to  keep  pace  with  this  alka- 
line production.  When  "ripe,"  0.4  per  cent,  of  trikresol 
is  added  to  the  cultures,  which  are  then  filtered  through 
porcelain.  If  the  toxin  must  be  kept  before  using,  it  is 
best  to  preserve  it  unfiltered,  as  it  deteriorates  more  rap- 
idly after  filtration.  Unfiltered  toxin  causes  too  much 
local  irritation.  If  the  bacillus  employed  was  virulent 
and  the  conditions  of  culture  were  favorable,  the  filtered 
culture  should  be  so  toxic  that  o.  i  c.cm.  would  be  fatal  to 
a  5OO-gram  guinea-pig  in  twenty-four  hours  (Roux).  Even 
under  the  most  favorable  circumstances  it  is  difficult  to 
obtain  a  toxin  which  will  kill  in  less  than  thirty  hours. 

The  experience  of  the  author  with  Fernbach' s  appara- 
tus has  not  been  satisfactory.  The  passing  current  of  air 
is  a  frequent  source  of  contamination  to  the  culture,  and 
requires  great  care.  In  the  end  it  is  questionable  whether 
the  toxin  thus  produced  is  better  than  that  obtained  from 
an  ordinary  flask  exposing  a  large  surface  to  the  air. 

Park  and  Williams  did  an  elaborate  work  upon  the 
production  of  diphtheria  toxin.1  They  found  that 
4 'toxin  of  sufficient  strength  to  kill  a  4oo-gram  guinea- 
pig  in  three  days  and  a  half  in  a  dose  of  0.025  c.cm., 
developed  in  suitable  bouillon,  contained  in  ordinary 
Erlenmeycr  flasks,  within  a  period  of  twenty-four  hours, 
•ich  bouillon  the  toxin  reached  its  greatest  strength 
in  four  to  seven  days  (0.005  c.cm.  killing  a  5oo-gram 

1  Jour,  of  Exper.  Med.t  vol.  i.,  No.  I,  Jan.,  1896,  p.  164. 


DIPHTHERIA.  299 

guinea-pig  in  three  days).  This  period  of  time  covered 
that  of  the  greatest  growth  of  the  bacilli,  as  shown  both 
by  the  appearance  of  the  culture  and  by  the  number  of 
colonies  developing  on  agar  plates." 

"The  bodies  of  the  diphtheria  bacilli  did  not  at  any 
time  contain  toxin  in  considerable  amounts."  "The 
type  of  growth  of  the  bacilli  and  the  rapidity  and  extent 
of  the  production  of  toxin  depended  more  on  the  reaction 
of  the  bouillon  than  upon  any  other  single  factor." 
"  The  best  results  were  obtained  in  bouillon  which,  after 
being  neutralized  to  litmus,  had  about  7  c.cm.  of  normal 
soda  solution  added  to  each  liter.  An  excessive  amount 
of  either  acid  or  alkali  prevented  the  development  of 
toxin."  "Strong  toxin  was  produced  in  bouillon  con- 
taining peptone  ranging  from  i  to  10  per  cent."  "The 
strength  of  toxin  averaged  greater  in  the  2  and  4  per 
cent,  peptone  solution  than  in  the  i  per  cent." 

"When  the  stage  of  acid  reaction  was  brief  and  the 
degree  of  acidity  probably  slight,  strong  toxin  developed 
while  the  culture  bouillon  was  still  acid;  but  when  the 
stage  of  acid  reaction  was  prolonged  little  if  any  toxin 
was  produced  until  just  before  the  fluid  became  alka- 
line." 

"Glucose  is  deleterious  to  the  growth  of  the  diphtheria 
bacillus  and  to  the  production  of  toxin  when  it  is  present 
in  sufficient  amounts  to  cause  by  its  disintegration  too 
great  a  degree  of  acidity  in  the  culture-fluid.  When  the 
acid  resulting  from  the  decomposition  of  glucose  is  neu- 
tralized by  the  addition  of  an  alkali  the  diphtheria 
bacillus  again  grows  abundantly." 

The  Immunization  of  the  Animal. — The  animals  chosen 
to  furnish  the  antitoxic  serum  should  be  animals  which 
present  a  distinct  natural  immunity  to  ordinary  doses  of 
the  toxin,  and  should  be  sufficiently  large  to  furnish  large 
quantities  of  the  finished  serum.  Behring  originally 
employed  dogs  and  sheep  ;  Aronson  at  first  preferred  the 
goat ;  but  Roux  introduced  the  horse,  which  is  more  easi- 
ly immunized  than  the  other  animals  mentioned,  and, 


300  PATHOGENIC  BACTERIA. 

being  large  enough  to  furnish  a  considerable  quantity 
of  serum,  recommends  itself  strongly  for  the  purpose. 

The  animal  chosen  should  be  free  from  tuberculosis 
and  glanders,  as  tested  by  tuberculin  and  mallein,  but 
need  not  be  expensive.  A  horse  with  a  disabled  foot 
will  answer  well.  Rheumatic  horses  should  be  rejected. 
In  the  beginning  a  small  dose  of  the  toxin — about  i 
c.cm. — should  be  given  hypodermically  to  detect  indi- 
vidual susceptibility.  Horses  vary  much  in  this  particu- 
lar, as  Roux  has  pointed  out.  The  author  found  light- 
colored  horses  to  be  distinctly  more  susceptible  than 
dark-colored  ones,  a  fact  which  has  some  substantiation 
in  the  clinical  observation  that  blonde  children  suffer 
more  severely  from  diphtheria  than  dark-complexioned 
ones. 

If  well  borne,  the  preliminary  injection  is  followed  in 
about  six  days  by  a  larger  dose,  in  six  days  more  by 
a  still  larger  one,  and  the  increase  is  continued  every  six 
days  or  so,  according  to  the  condition  of  the  animal, 
until  enormous  quantities — 500-1,000  c.cm. — are  intro- 
duced at  a  time. 

As  the  expression  of  quantity  alone  is  very  misleading, 
and  to  know  exactly  what  strength  the  horse  is  receiving, 
the  author  has  devised  a  special  nomenclature  by  which 
to  express  it.  Instead  of  stating  that  the  animal  received 
10,  50,  or  loo  c.cm.  of  toxin,  we  now  say  it  receives  10, 
50,  or  loo  factors,  the  term  factor  being  used  to  express 
loo  times  the  least  certainly  fatal  dose  of  toxin  per  100 
grams  of  guinea-pig.  The  number  of  factors  in  a  given 
quantity  of  antitoxin  naturally  varies  with  its  strength, 
and  it  will  at  once  be  seen  that  it  is  advantageous  to  ex- 
press strength  regardless  of  quantity. 

The  toxin  causes  some  local  reaction — at  first  a  dis- 
tinct inflammation,  later  a  painful  edema  and  a  febrile 
reaction.  The  amount  of  local  irritation  is  much  less 
marked  when  the  injections  are  made  slowly ;  and  a 
gravity  apparatus,  which  is  filled  with  the  amount  of 
scrum  to  be  injected,  suspended  from  the  ceiling  of  the 


DIPHTHERIA.  301 

stable  so  that  the  toxin  is  allowed  to  take  its  own  time 
to  enter  the  tissues,  can  be  recommended.  Sometimes 
it  takes  an  hour  to  inject  500  c.cm.  in  this  manner. 

The  amount  of  local  reaction,  edema,  etc.,  the  appetite 
and  general  condition,  the  temperature-curve,  and  the 
stability  of  the  body-weight,  must  all  be  taken  into  con- 
sideration, so  that  the  administration  shall  not  be  too 
rapid  and  the  animal  be  thrown  into  a  condition  of 
cachexia  with  toxic  instead  of  antitoxic  blood. 

One  of  the  principal  things  to  be  avoided  is  haste. 
Too  frequent  or  too  large  dosage  is  almost  certain  to  kill 
the  animal. 

Behring  found  that  mixing  the  toxin  with  trichlorid 
of  iodin  lessened  the  irritant  effect  upon  susceptible  ani- 
mals. I  prefer  not  to  use  susceptible  horses. 

The  suggestion  of  Prof.  Pearson,  that  the  large  doses 
of  toxin  might  with  readiness  be  introduced  into  the 
trachea  when  the  absorption  is  good,  has  been  success- 
fully accomplished  by  the  author.  The  absorption  seems 
to  take  place  without  any  change  in  the  toxin,  and  to  be 
as  rapid  as  from  the  subcutaneous  tissue. 

As  the  antitoxin  protects  the  horse  perfectly  against 
the  toxin,  a  preliminary  dose  will  enable  one  to  omit  all 
the  small  preliminary  doses  of  toxin,  and  render  the 
horse  immune  at  once.  Thus,  I  have  frequently  adminis- 
tered 100  c.cm.  of  antitoxin  of  about  100  units  strength 
to  a  horse  one  day  and  500  c.cm.  of  strong  toxin  (500 
factors)  the  next.  This  is  just  500  times  as  much  toxin 
as  has  twice  killed  a  horse  in  the  laboratory.  After  the 
lapse  of  a  few  days  the  same  quantity  can  be  administered 
again,  and  in  a  week  a  third  time.  In  this  rapid  way 
antitoxin  can  often  be  secured  at  short  notice.  It  is  yet 
a  question,  however,  whether  this  method,  modified  from 
Pawlowski,  is  as  good  and  certain  as  the  slow  way  sug- 
gested by  Behring. 

The  possibility  of  producing  serum  rapidly  may  depend 
upon  the  method,  but  the  production  of  strong  serums  de- 
pends chiefly  upon  the  horse  and  not  upon  its  treatment. 


302  PATHOGENIC  BACTERIA. 

The  Preparation  of  the  Serum  for  Therapeutic  Pztr- 
floses.—When,  because  of  the  tolerance  to  large  quanti- 
ties of  toxin,  the  horse  seems  to  possess  antitoxic  blood, 
a  "twitch"  is  applied  to  the  upper  lip,  the  eyes  are 
blindfolded,  a  small  incision  is  made  through  the  skin, 
a  trocar  thrust  into  the  jugular  vein,  and  the  blood  al- 
lowed to  flow  through  a  cannulated  tube  into  sterile 
bottles.  It  is  allowed  to  coagulate,  and  kept  upon  ice 
for  two  days  or  so,  that  the  clear  serum  may  be  pi- 
petted off.  This  serum  is  the  antitoxic  serum.  It  does 
not  always  materialize  according  to  the  desires  of  the 
experimenter,  sometimes  proving  surprisingly  strong  in 
a  short  time,  sometimes  very  weak  after  months  of 
patient  preparation. 

The  serums  are  preserved  by  Roux  with  camphor,  by 
Behring  with  carbolic  acid  (0.5  per  cent.),  and  by  Aron- 
son  with  trikresol  (0.4  per  cent.).  I  prefer  to  use  tri- 
kresol,  as  it  is  not  poisonous,  is  a  reliable  antiseptic,  and 
has  a  very  pronounced  local  anesthetic  action.  Formalin 
has  been  tried,  but  it  gelatinizes  the  serum  and  causes 
much  local  pain  when  injected  beneath  the  skin. 

Dried  antitoxic  serum  has  also  been  placed  upon  the 
market  under  the  impression  that  it  will  keep  longer  and 
bear  shipment  better  than  any  other.  This  is  not,  how- 
ever, shown  to  be  the  case,  and  as  the  dried  serum  dis- 
solves with  difficulty  it  is  much  less  convenient  than  the 
usual  preparations.  It  is  also  less  likely  to  be  sterile 
than  the  liquid  forms. 

The  strength  of  the  serum  is  expressed  in  what  are 
known  as  immunizing  units.  This  denomination  origin- 
ated with  Behring  and  Ehrlich,  whose  normal  serum 
was  of  such  strength  that  o.i  c.cm.  of  it  would  protect 
against  ten  times  the  least  certainly  fatal  dose  of  toxin 
when  simultaneously  injected  into  guinea-pigs.  Each 
cubic  centimeter  of  this  normal  serum  they  called  an 
immunizing  unit.  Later  it  was  shown  that  the  strength 
of  the  serum  could  easily  be  increased  tenfold,  so  that 
o.oi  c.cm.  of  the  serum  would  protect  the  guinea-pig 


DIPHTHERIA.  303 

against  the  ten-times  fatal  dose.  Each  cubic  centimeter 
of  this  stronger  serum  was  described  as  an  antitoxic  unit, 
and,  of  course,  contained  ten  immunizing  units.  Still 
later  it  was  shown  that  the  limits  of  strength  were  by 
no  means  reached,  and  he  succeeded  in  making  serums 
three  hundred  times  the  normal  strength,  each  cubic 
centimeter  of  which  contained  300  immunizing  units, 
or  30  antitoxic  units. 

In  the  course  of  the  development  of  strength  in  the 
serum  the  exact  meaning  of  u  immunizing  unit "  grad- 
ually became  obscured,  until  it  is  at  present  an  expres- 
sion of  strength  rather  than  one  of  quantity. 

While  it  is  difficult  to  define  an  immunizing  unit,  it  is 
not  at  all  difficult  for  one  skilled  in  laboratory  technique 
to  determine  the  number  present  in  a  sample  of  serum. 
There  are  three  rules  of  practice: 

1.  Determine  accurately  the  least  certainly  fatal  dose 
of  a  sterile  diphtheria  toxin  for  a  standard  guinea-pig. 

2.  Determine    accurately   the    least    quantity   of   the 
serum  that  will  protect  a  guinea-pig  against  ten  times 
the  determined  least  certainly  fatal  dose  of  toxin. 

3.  Express  the  required  dose  of  antitoxic  serum  as  a 
fraction  of  a  cubic  centimeter  and  multiply  it  by  ten. 
There  will  then  be  as  many  immunizing  units  in  i  c.cm. 
of  the  serum  as  there  are  parts  in  the  resulting  fraction. 

Example:  It  is  found  that  o.oi  c.cm.  of  toxin  kills  at 
least  9  out  of  10  guinea-pigs.  It  is  then  regarded  as  the 
least  certainly  fatal  dose.  Guinea-pigs  receive  ten 
times  this  dose  (o. i  c.cm.)  and  varying  quantities  of  the 
serum,  measured  by  dilution,  say,  y^Vir  c.cm.,  y^nr  c.cm., 
ygVfr  c-cm-  The  first  two  live.  The  fraction  y^^nr  is 
now  multiplied  by  10;  ^oW  X  10  =  2"3T>  an(^  we  find  that 
there  are  as  many  units  per  cubic  centimeter  in  the  serum 
as  there  are  parts  in  the  result — i.  <?.,  250. 

The  most  accurate  definition  of  an  immunizing  unit  is: 
ten  times  the  least  amount  of  antitoxic  serum  that  will 
protect  a  standard  (3OO-gram)  guinea-pig  against  ten 
times  the  least  certainly  fatal  dose  of  diphtheria  toxin. 


304  PA  THOGENIC  BA  CTERIA . 

The  strongest  serum  ever  obtained  by  the  author  con- 
tained 1400  units  per  cubic  centimeter. 

As  the  quantity  to  be  injected  at  each  dose  diminishes 
according  to  the  number  of  units  per  cubic  centimeter 
the  serum  contains,  it  is  of  the  highest  importance  that 
the  serums  be  as  strong  as  possible.  Various  methods  of 
concentration  have  been  suggested,  such  as  the  partial 
evaporation  of  the  serum  in  vacuo,  but  none  have 
proved  satisfactory.  The  latest  suggestion  comes  from 
Bujwid,1  who  finds  that  when  an  antitoxic  serum  is 
frozen  and  then  thawed,  it  separates  into  two  layers, 
an  upper  watery  stratum  and  a  lower  yellowish  one;  the 
antitoxic  value  of  the  yellowish  layer  is  about  three 
times  that  of  the  original  serum. 

Ehrlich  asserts  that  500  units  are  valueless:  2000  units 
are  probably  an  average  dose,  and,  as  the  remedy  seems 
harmless,  it  is  better  to  err  on  the  side  of  too  much  than 
on  that  of  too  little.  Fourteen  thousand  units  have  been 
administered  in  one  case  with  beneficial  results. 

The  largest  collection  of  statistics  upon  the  results  of 
antitoxic  treatment  in  diphtheria  in  the  hospitals  of  the 
world  are  probably  those  collected  by  Prof.  Welch,  who, 
excluding  every  possible  error  in  the  calculations,  "shows 
an  apparent  reduction  of  case-mortality  of  55.8  per  cent." 
One  of  the  most  important  things  in  the  treatment  is 
to  begin  it  early  enough.  Welch's  statistics  show  that 
1115  cases  of  diphtheria  treated  in  the  first  three  days 
of  the  disease  yielded  a  fatality  of  8.5  per  cent,  whereas 
546  cases  in  which  the  antitoxin  was  first  injected  after 
the  third  day  of  the  disease  yielded  a  fatality  of  27.8  per 
cent. 

After  the  toxin  has  set  up  destructive  organic  lesions 
in  various  organs  and  tissues  of  the  body,  no  amount 
of  neutralization  will  restore  the  integrity  of  the  parts, 
so  that  the  antitoxin  must  fail  in  these  cases. 

The  urticaria  which  sometimes  follows  the  injection 

1  Centralbl.  f.  Bakt.  u.  Parasitenk.,  Sept.,  1897,  Bd.  xxii.,  Nos.  10  and  II, 
p.  287. 


DIPHTHERIA.  305 

of  antitoxic  serum  seems  to  bear  a  distinct  relation  to 
the  age  of  the  serum,  fresh  serums  being  more  liable 
to  produce  it  than  those  which  have^been  kept  for  a 
month  or  two. 

I  have  found  that  the  "keeping"  qualities  of  the  se- 
rums, when  properly  preserved,  are  of  long  durati6n. 
Samples  examined  two  years  after  having  been  exposed 
for  sale  in  the  markets  have  been  found  unchanged. 
The  serums  most  prone  to  deteriorate  seem  to  be  those 
of  highest  potency,  but  even  here  the  good  qualities  are 
unchanged  for  months. 

Freezing  is  without  effect  and  ordinary  temperature- 
changes  are  harmless  to  the  serum.  The  antitoxic  power 
is  destroyed  at  72°  C.,  the  point  at  which  the  serum 
coagulates. 

The  erythemata  are  probably  in  some  way  associated 
with  the  globulicidal  action  of  the  blood.  Keeping  the 
serum  "  until  it  is  ripe  "  lessens  this  effect.  The  serums 
from  different  horses  probably  vary  much  in  both  their 
irritant  and  globulicidal  properties,  so  that  antitoxins 
prepared  by  mixing  the  serums  from  a  number  of  horses 
are  probably  preferable  to  those  from  single  horses. 

Dried  serums  are  much  less  active  than  fresh  ones. 

For  purposes  of  immunization  smaller  doses  than  those 
used  for  treatment  suffice.  According  to  Biggs,  2  cubic 
centimeters  are  sufficient  to  give  complete  protection. 
The  immunity  that  results  from  the  injection  is  of  a 
month  or  six  weeks'  duration. 

The  transitory  nature  of  this  immunity  is  probably 
dependent  upon  the  fact  that  the  antitoxin  is  slowly  ex- 
creted through  the  kidneys. 

20 


CHAPTER    III. 
HYDROPHOBIA,  OR  RABIES. 

No  micro-organism  of  hydrophobia  has  as  yet  been 
discovered,  yet  the  peculiarities  of  the  disease  are  such 
as  to  leave  no  doubt  in  the  mind  of  a  bacteriologist  that 
one  must  exist.  To  find  it  is  now  the  desideratum. 

Although  many  men  have  labored  upon  hydrophobia, 
no  name  is  so  well  known  or  so  justly  honored  as  that 
of  the  great  pioneer  in  bacteriology,  Pasteur.  The  profes- 
sion and  laity  are  alike  familiar  with  his  name  and  work, 
and  although  at  times  the  newspapers  of  our  country 
and  certain  members  of  the  profession  have  opposed  the 
methods  of  treatment  which  he  has  suggested  as  the  re- 
sult of  his  experimentation,  we  cannot  but  feel  that  this 
skepticism  and  opposition  are  due  either  to  ignorance 
of  the  principles  upon  which  Pasteur  reasoned  or  to  a 
culpable  conservatism.  The  most  vehement  opponent 
that  Pasteur  has  in  America  seems  to  disbelieve  the 
existence  of  rabies.  It  is  impossible  to  argue  with  him. 

Hydrophobia,  or  rabies,  is  a  specific  toxemia  to  which 
dogs,  wolves,  skunks,  and  cats  are  highly  susceptible, 
and  which  can,  through  their  saliva,  be  communicated 
to  men,  horses,  cows,  and  other  animals.  The  means 
of  communication  is  almost  invariably  a  bite,  hence  the 
inference  that  the  specific  organism  is  present  in  the 
saliva. 

The  animals  that  are  infected  manifest  no  symptoms 
(luring  a  varying  incubation-period  in  which  the  wound 
generally  heals  kindly.  This  period  may  last  for  as  long 
a  time  as  twelve  months,  but  in  rare  cases  may  be  only 
some  days.  An  average  duration  of  the  period  of  incu- 
bation might  be  stated  as  about  six  weeks. 

306 


HYDROPHOBIA,  OR  RABIES.  307 

As  the  incubation-period  comes  to  an  end  there  is  an 
observable  alteration  in  the  wound,  which  becomes  red- 
dened, sometimes  may  suppurate  a  little,  and  is  painful. 
The  victim,  if  a  man,  is  much  alarmed  and  has  a  sensa- 
tion of  horrible  dread.  The  period  of  dread  passes  into 
one  of  excitement,  which  in  many  cases  amounts  to  a 
wild  delirium  and  ends  in  a  final  stage  of  convulsion  and 
palsy.  The  convulsions  are  tonic,  rarely  clonic,  and 
subsequently  cause  death  by  interfering  with  the  respira- 
tion, as  do  those  of  tetanus  and  strychnia. 

During  the  convulsive  period  much  difficulty  is  experi- 
enced in  swallowing  liquids,  and  it  is  supposed  that  the 
popular  term  "hydrophobia"  arose  from  the  reluctance 
of  the  diseased  to  take  water  because  of  the  inconveni- 
ence and  occasional  spasms  which  the  attempt  causes. 

This  description,  brief  and  imperfect  as  it  is,  will 
illustrate  the  parallelism  existing  between  hydrophobia 
and  tetanus.  In  the  latter  we  observe  the  entrance  of 
infectious  material  through  a  wound,  which,  like  the 
bite  in  hydrophobia,  sometimes  heals,  but  often  suppu- 
rates a  little.  We  see  in  both  affections  an  incubation- 
period  of  varying  duration,  though  in  hydrophobia  it  is 
much  longer  than  in  tetanus,  and  convulsions  of  tonic 
character  causing  death  by  asphyxia. 

It  is  maintained  by  some  that  the  stage  of  excitement 
argues  against  the  specific  nature  of  the  disease,  but 
these  subjective  symptoms  are  like  the  mental  con- 
dition of  tuberculosis,  which  leads  the  patient  to  make  a 
hopeful  prognosis  of  his  case,  and  the  mental  condition 
of  anthrax,  in  which  it  is  stated  that  no  matter  how  dan- 
gerous his  condition  the  patient  is  seldom  much  alarmed 
about  it. 

Pasteur  and  his  co-workers  found  that  in  animals  that 
die  of  rabies  the  salivary  glands,  the  pancreas,  and  the 
nervous  system  contain  the  infection,  and  are  more 
appropriate  for  experimental  purposes  than  the  saliva, 
which  is  invariably  contaminated  with  accidental  patho- 
genic bacteria. 


308  PA  THOGENIC  BA  CTERIA. 

The  introduction  of  a  fragment  of  the  medulla  ob- 
longata  of  a  dog  dead  of  rabies  beneath  the  dura  mater 
of  a  rabbit  causes  the  development  of  rabies  in  the 
rabbit  in  a  couple  of  weeks.  The  medulla  of  this  rabbit 
introduced  beneath  the  dura  mater  of  a  second  rabbit 
produced  a  more  violent  form  of  the  disease  in  a  shorter 
time,  and  by  frequently  repeated  implantations  Pasteur 
found  that  an  extremely  virulent  material  could  be  ob- 
tained. 

Inasmuch  as  the  toxins  of  diphtheria  and  tetanus 
circulate  in  the  blood,  and  not  infrequently  saturate 
the  nervous  systems  of  animals  affected,  it  might  be 
concluded  that  the  material  with  which  Pasteur  worked 
was  a  toxin.  This  is  readily  disproven,  however,  not 
only  by  the  fact  that  the  toxin  would  weaken  instead  of 
strengthen  by  the  method  of  transfer  from  animal  to 
animal,  it  not  being  a  vital  entity,  but  also  by  the  dis- 
covery that  when  an  emulsion  of  the  nervous  system  of 
an  affected  animal  is  filtered  through  porcelain,  or  when 
it  is  heated  for  a  few  moments  to  100°  C.,  or  exposed 
for  a  considerable  time  to  a  temperature  of  75°  or  80°  C., 
its  virulence  is  entirely  lost.  This  would  seem  to  prove 
that  that  which  is  in  the  nervous  system  and  communi- 
cates the  disease  is  a  living,  active  body — a  parasite,  and 
in  all  probability  a  bacterium.  However,  all  endeavors 
to  discover,  isolate,  or  cultivate  this  organism  have  failed. 

Pasteur  noted  that  the  virulence  of  the  poison  was  less 
in  animals  that  had  been  dead  for  some  time  than  in 
the  nervous  systems  of  those  just  killed,  and  by  experi- 
mentation showed  that  when  the  nervous  system  was 
dried  in  a  sterile  atmosphere  the  virulence  was  attenu- 
ated in  proportion  to  the  length  of  time  it  had  been  dry. 
This  attenuation  of  virulence  of  course  suggested  to 
Pasteur  the  idea  of  a  protective  vaccination,  and  by  in- 
oculating a  dog  with  much  attenuated,  then  with  less 
attenuated,  then  with  moderately  strong,  and  finally  with 
strong,  virus,  the  dog  developed  an  immunity  which 
enabled  it  to  resist  the  infection  of  an  amount  of  viru- 


HYDROPHOBIA,  OR  RABIES.  309 

lent  material  that  would  certainly  kill  an  unprotected 
animal. 

It  is  remarkable  that  this  thought,  which  was  a  theory 
based  upon  a  broad  knowledge,  but  experience  with 
comparatively  few  bacteria,  should  every  day  find  more 
and  more  grounds  for  confirmation  as  our  knowledge 
of  immunity,  of  toxins,  and  of  antitoxins  progresses. 
What  Pasteur  did  with  rabies  is  what  we  now  do  in 
producing  the  antitoxin  of  diphtheria — i.  e.  gradually 
accommodate  the  animal  to  the  poison  until  its  body-cells 
are  able  to  neutralize  or  resist  it.  As  the  poison  cannot 
be  secured  outside  of  the  body  because  the  bacilli,  micro- 
cocci,  or  whatever  they  may  be  cannot  be  secured  outside 
of  the  body,  he  does  what  Behring  originally  did  in  diph- 
theria— introduces  attenuated  poison-producers — bacilli 
crippled  by  heat  or  drying,  and  capable  of  producing  only 
a  little  poison — accustoms  the  animal  to  these,  and  then  to 
stronger  and  stronger  ones  until  immunity  is  established. 

The  genius  of  Pasteur  did  not  cease  with  the  produc- 
tion of  immunity,  but,  we  rejoice  to  add,  extended  to  the 
kindred  subject  of  therapy,  and  has  now  given  us  a  cure 
for  hydrophobia. 

For  the  production  of  a  cure  in  infected  cases  very 
much  the  same  treatment  is  followed  as  has  been  de- 
scribed for  the  production  of  immunity.  The  patient 
must  come  under  observation  early.  The  treatment  con- 
sists of  the  subcutaneous  injection  of  about  2  grams  of 
an  emulsion  of  a  rabbit's  spinal  cord  which  had  been 
dried  for  from  seven  to  ten  days.  This  beginning  dose 
is  not  increased  in  size,  but  each  day  the  emulsion  used 
is  from  a  cord  which  has  not  been  dried  so  long,  until, 
when  the  twenty-fifth  day  of  treatment  is  reached,  the 
patient  receives  2  grams  of  emulsion  of  spinal  cord  dried 
only  three  days,  and  is  considered  immune  or  cured. 

It  will  be  observed  that  this  treatment  is  really  no 
more  than  the  immunization  of  the  individual  during  the 
incubation  stadium,  and  the  generation  of  a  vital  force — 
shall  we  call  it  an  antitoxin  ? — in  the  blood  of  the  animal 


310  PATHOGENIC  BACTERIA. 

in  advance  of  the  time  when  the  organism  is  expected  to 
saturate  the  body  with  its  toxic  products. 

This,  in  brief,  is  the  theory  and  practice  of  Pasteur's 
system  of  treating  hydrophobia.  It  is  exactly  in  keeping 
with  the  ideas  of  the  present,  and  is  most  extraordinary 
in  its  reasonings  and  details  when  we  remember  that  the 
first  application  of  the  method  to  human  medicine  was 
made  October  26,  1885,  nearly  ten  years  before  the  time 
we  began  to  understand  the  production  and  use  of  anti- 
toxins. 


CHAPTER   IV. 

CHOLERA  AND  SPIRILLA  RESEMBLING  THE  CHOLERA 
SPIRILLUM. 

CHOLERA  is  a  disease  from  which  certain  parts  of  India 
are  never  free.  The  areas  in  which  it  is  endemic  are 
the  foci  from  which  the  great  epidemics  of  the  world,  as 
well  as  the  constant  smaller  epidemics  of  India,  probably 
spread.  No  one  knows  when  cholera  was  first  introduced 
into  India,  and  the  probabilities  are  that  it  is  indigenous 
to  that  country,  as  yellow  fever  is  to  Cuba.  Very  early 
mention  of  it  is  made  in  the  letters  of  travellers,  in 
books  and  papers  on  medicine  of  a  century  ago,  and 
in  the  governmental  statistics,  yet  we  find  that  little  is 
said  about  the  disease  except  in  a  general  way,  most 
attention  being  directed  to  the  effect  upon  the  armies, 
native  and  European,  of  India  and  adjacent  countries. 
The  opening  up  of  India  by  Great  Britain  in  the  last 
half  century  has  made  possible  much  accurate  scientific 
observation  of  the  disease  and  the  relation  which  its  epi- 
demics bear  to  the  manners  and  customs  of  the  people. 

The  filthy  habits  of  the  people  of  India,  their  poverty, 
their  crowded  condition,  and  their  religious  customs,  all 
serve  to  aid  in  the  distribution  of  the  disease.  We  are 
told  that  the  city  of  Benares  drains  into  the  Ganges  River 
by  a  most  imperfect  system,  which  distributes  the  greater 
part  of  the  sewage  immediately  below  the  banks  upon 
which  the  city  is  built.  It  is  a  matter  of  religious  ob- 
servance for  every  zealot  who  makes  a  pilgrimage  to  the 
1 '  sacred  city  ' '  to  take  a  bath  in  and  drink  a  large  quan- 
tity of  this  sacred  but  polluted  water,  and,  as  may  be 
imagined,  the  number  of  pious  Hindoos  who  leave 
Benares  with  comma  bacilli  in  their  intestines  or  upon 
their  clothes  is  great,  for  there  are  few  months  in  the 

311 


312  PATHOGENIC  BACTERIA. 

year  when  there  are  not  at  least  some  cases  of  cholera 
in  the  city. 

The  frequent  pilgrimages  and  great  festivals  of  the 
Hindoos  and  Moslems,  by  bringing  together  an  enormous 
number  of  people  who  crowd  in  close  quarters  where  filth 
and  bad  diet  are  common,  cause  a  rapid  increase  in  the 
number  of  cases  during  these  periods  and  the  dispersion 
of  the  disease  when  the  festivals  break  up.  The  disease 
extends  readily  along  the  regular  lines  of  travel,  visiting 
town  after  town,  until  from  Asia  it  has  frequently  ex- 
tended into  Europe,  and  by  the  steamships  plying  on 
foreign  waters  has  been  several  times  carried  to  our  own 
continent  and  to  the  islands  of  the  seas.  Many  cases  are 
on  record  which  show  conclusively  how  a  single  ship, 
having  a  few  cholera  cases  on  board,  may  be  the  cause 
of  an  outbreak  of  the  disease  in  the  port  at  which  it 
arrives. 

It  seems  strange  to  us  now,  with  the  light  of  present 
information  illuminating  the  pages  of  the  past,  to  observe 
how  the  distinctly  infectious  nature  of  such  a  disease 
could  be  overlooked  in  the  search  for  some  atmospheric 
or  climatic  cause,  some  miasm,  which  was  to  account 
for  it. 

The  discovery  of  the  organism  which  seems  to  be  the 
specific  cause  of  cholera  was  made  by  Koch,  who  was 
appointed  one  of  a  German  cholera-commission  to  study 
the  disease  in  Egypt  and  India  in  1883-84.  Since  his 
discovery,  but  a  decade  ago,  the  works  upon  cholera  and 
the  published  investigations  to  which  the  spirillum  has 
been  subjected  have  produced  an  immense  literature, 
a  large  part  of  which  was  stimulated  by  the  Hamburg 
epidemic  of  a  few  years  ago. 

The  micro-organism  described  by  Koch,  and  now  gen- 
erally accepted  to  be  the  cause  of  cholera,  is  a  short 
individual  about  half  the  length  of  a  tubercle  bacillus, 
considerably  stouter,  and  distinctly  curved,  so  that  the 
original  name  by  which  it  was  known  was  the  "comma 
bacillus"  (Figs.  80,  81). 


CHOLERA.  313 

A  study  of  the  growth  of  the  organism  and  the  forms 
which  it  assumes  upon  different  culture-media  soon  con- 
vinces us  that  we  have  to  do  with  an  organism  in  no  way 
related  to  the  bacilli.  If  the  conditions  of  nutrition  are 


.'7  ,., 


FIG.  80. — Spirillum  of  Asiatic  cholera,  showing  the  flagella;  x  1000  (Gunther). 

diminished  so  that  the  multiplication  of  the  bacteria  by 
simple  division  does  not  progress  with  the  usual  rapidity, 
we  find  a  distinct  tendency  toward — and  in  some  cases, 
as  upon  potato,  a  luxuriant  development  of — long  spiral 
threads  with  numerous  windings — unmistakable  spirilla. 
Frankel  has  found  that  the  exposure  of  cultures  to  unusu- 
ally high  temperatures,  the  addition  of  small  amounts 
of  alcohol  to  the  culture-media,  etc.,  will  so  vary  the 
growth  of  the  organism  as  to  favor  the  production  of 
spirals  instead  of  commas.  One  of  the  most  common 
of  the  numerous  forms  observed  is  that  in  which  two 
short  curved  individuals  are  so  joined  as  to  produce  an 
S-shaped  curve. 

The  cholera  spirilla  are  exceedingly  active  in  their 
movements,  and  in  hanging-drop  cultures  can  be  seen 
to  swim  about  with  great  rapidity.  Not  only  do  the 
comma-shaped  organisms  move,  but  when  distinct  spirals 
exist,  they,  too,  move  with  the  rapid  rotary  motion  so 
common  among  the  spirilla. 


314  PATHOGENIC  BACTERIA. 

The  presence  of  flagella  upon  the  cholera  spirillum 
can  be  demonstrated  without  difficulty  by  Loffler's 
method  (q.  z/.).  Each  spirillum  possesses  a  single  flagel- 
lum  attached  to  one  end. 

Inoculation-forms  of  most  bizarre  appearance  are  very 
common  in  old  cultures  of  the  spirillum,  and  very  often 


i    v  \-*>  .    y&  ;  -r* ; 


C^V  <  <^..\ 

^    At.'     |^-         ^(i-V     i^t-       * 

V  r  -•«»  ,()     1 


3^ 


FlG.  8l. — Spirillum  of  Asiatic  cholera,  from  a  bouillon  culture  three  weeks  old, 
showing  numbers  of  long  spirals;    x  1000  (Frankel  and  Pfeiffer). 

there  can  be  found  in  fresh  cultures  many  individuals 
which  show  by  granular  protoplasm  and  irregular  outline 
that  they  are  partly  degenerated.  Cholera  spirilla  from 
various  sources  seem  to  differ  in  this  particular,  some 
of  the  forms  being  as  pronounced  in  their  involution 
as  the  diphtheria  bacilli. 

In  partially  degenerated  cultures  in  which  long  spirals 
are  numerous  Hiippe  observed,  by  examination  in  the 
" hanging  drop,"  in  the  continuity  of  the  elongate  mem- 
bers, certain  large  spherical  bodies  which  he  described  as 
spores.  These  bodies  were  not  enclosed  in  the  organisms 
like  the  spores  of  anthrax,  but  seemed  to  exemplify  the 
form  of  sporulation  in  which  an  entire  individual  trans- 
forms itself  into  a  spore  (arthrospore).  Koch,  and  indeed 
all  other  observers,  failed  to  find  signs  of  fructification  in 


CHOLERA.  315 

the  cholera  organism,  and  the  true  nature  of  the  bodies 
described  by  Hiippe  must  be  regarded  as  doubtful. 
Most  bacteriologists  disagree  with  Hiippe  in  believing 
that  arthrospores  exist  at  all,  and  the  fact  (which  will  be 
pointed  out  later  on)  that  there  is  very  little  permanence 
about  cholera  cultures  throws  additional  doubt  upon  the 
accuracy  of  Hiippe' s  conclusion. 

The  cholera  spirillum  stains  well  with  the  ordinary 
aqueous  solutions  of  the  anilin  dyes  ;  fuchsin  seems  par- 
ticularly appropriate.  At  times  the  staining  must  be  con- 
tinued for  from  five  to  ten  minutes  to  secure  homogeneity. 
The  cholera  spirillum  does  not  stain  by  Gram's  method. 
It  may  be  colored  and  examined  while  alive ;  thus  Cornil 
and  Babes,  in  demonstrating  it  in  the  rice-water  dis- 
charges, "spread  out  one  of  the  white  mucous  fragments 
upon  a  glass  slide  and  allow  it  to  dry  partially  ;  a  small 
quantity  of  an  exceedingly  weak  solution  of  methyl  violet 
in  distilled  water  is  then  flowed  over  it,  and  it  is  flattened 
out  by  pressing  down  on  it  a  cover-glass,  over  which  is 
placed  a  fragment  of  filter-paper,  which  absorbs  any 
excess  of  fluid  at  the  margin  of  the  cover-glass.  Comma 
bacilli  so  prepared  and  examined  with  an  oil-immersion 
lens  (x  700-800)  may  then  be  seen :  their  characters  are 
the  more  readily  made  out  because  of  the  slight  stain 
which  they  take  up,  and  because  they  still  retain  their 
power  of  vigorous  movement,  which  would  be  entirely 
lost  if  the  specimen  were  dried,  stained,  and  mounted  in 
the  ordinary  fashion." 

The  colonies  of  the  spirillum  when  grown  upon  gel- 
atin plates  are  highly  characteristic.  They  appear  in 
the  lower  strata  of  the  gelatin  as  small  white  dots,  grad- 
ually grow  out  to  the  surface,  effect  a  gradual  liquefaction 
of  the  medium,  and  then  appear  to  be  situated  in  little 
pits  with  sloping  sides  (Fig.  82).  This  peculiar  appear- 
ance, which  gives  one  the  suggestion  that  the  plate  is 
full  of  little  holes  or  air-bubbles,  is  due  to  the  evapora- 
tion of  the  liquefied  gelatin. 

One  of  the  best  methods  of  securing  pure  cultures  of 


PATHOGENIC  BACTERIA 

the  cholera  spirillum,  and  also  of  making  a  diagnosis 
of  the  disease  in  a  suspected  case,  is  probably  that  of 
Schottelius.  The  method  is  very  simple :  A  small  quan- 
tity of  the  fecal  matter  is  mixed  with  bouillon  and  stood 
in  an  incubating  oven  for  twenty-four  hours.  If  the 


FIG.  82. — Spirillum  of  Asiatic  cholera;  colonies  two  days  old  upon  a  gelatin 
plate;    x  35  (Heim). 

cholera  spirilla  are  present,  they  will  grow  most  rapidly 
at  the  surface  of  the  liquid  when  the  supply  of  air  is 
good.  A  pellicle  will  be  formed,  a  drop  from  which, 
diluted  in  melted  gelatin  and  poured  upon  plates,  will 
show  typical  colonies. 

Under  the  microscope  the  principal  characteristics 
can  be  made  out.  The  colony  of  the  cholera  spirillum 
scarcely  resembles  that  of  any  other  organism.  The  little 
colonies  which  have  not  yet  reached  the  surface  of  the 
gelatin  begin  very  soon  to  show  a  pale-yellow  color  and 
to  exhibit  irregularities  of  contour,  so  that  they  are 
almost  never  smooth  and  round.  They  are  coarsely 
granular,  and  have  the  largest  granules  in  the  centre. 
As  the  colony  increases  in  size  the  granules  also  increase 


CHOLERA.  317 

In  size,  and  attain  a  peculiar  transparent  character  which 
is  suggestive  of  powdered  glass.  The  commencement 
of  liquefaction  causes  the  colony  to  be  surrounded  with  a 
transparent  halo.  When  this  occurs  the  colony  begins  to 
sink,  from  the  digestion  and  evaporation  of  the  medium, 
and  also  to  take  on  a  peculiar  rosy  color. 

In  puncture-cultures  in  gelatin  the  growth  is  again  so 
characteristic  that  it  is  quite  diagnostic  (Fig.  83).     The 


FIG.  83. — Spirillum    cholera  Asiatica  ;    gelatin   puncture-cultures    aged   forty- 
eight  and  sixty  hours  (Shakespeare). 


growth  takes  place  along  the  entire  puncture,  but  devel- 
ops best  at  the  surface,  where  it  is  in  contact  with  the 
atmosphere.  An  almost  immediate  liquefaction  of  the 
medium  begins,  and,  keeping  pace  with  the  rapidity  of 
the  growth,  is  more  marked  at  the  surface  than  lower 
down.  The  result  of  this  is  the  occurrence  of  a  short, 
rather  wide  funnel  at  the  top  of  the  puncture.  As  the 
growth  continues  evaporation  of  the  medium  takes  place 
slowly,  so  that  the  liquefied  gelatin  is  lower  than  the 
solid  surrounding  portions,  and  appears  to  be  surmounted 
by  an  air-bubble. 


3i8  PATHOGENIC  BACTERIA. 

The  luxuriant  development  of  the  spirilla  in  gelatin 
produces  considerable  solid  material  to  sediment  and  fill 
up  the  lower  third  or  lower  half  of  the  liquefied  area. 
This  solid  material  consists  of  masses  of  spirilla  which 
have  probably  completed  their  life-cycle  and  become 
inactive.  Under  the  microscope  they  exhibit  the  most 
varied  involution-forms.  The  liquefaction  reaches  the 
sides  of  the  tube  in  from  five  to  seven  days.  Liquefac- 
tion of  the  medium  is  not  complete  for  several  weeks. 
According  to  Frankel,  in  eight  weeks  the  organisms  in 
the  liquefied  culture  all  die,  and  cannot  be  transplanted. 
Kitasato,  however,  has  found  them  living  and  active  on 
agar-agar  after  ten  to  thirty  days,  and  Koch  was  able 
to  demonstrate  their  vitality  after  two  years. 

When  planted  upon  the  surface  of  agar-agar  the  spi- 
rilla produce  a  white,  shining,  translucent  growth  along 
the  entire  line  of  inoculation.  It  is  in  no  way  peculiar. 
The  vitality  of  the  organism  is  retained  much  better  upon 
agar-agar  than  upon  gelatin,  and,  according  to  Frankel, 
the  organism  can  be  transplanted  and  grown  when  nine 
months  old. 

The  growth  upon  blood-serum  likewise  is  without  dis- 
tinct peculiarities,  and  causes  gradual  liquefaction  of  the 
medium. 

Upon  potato  the  spirilla  grow  well,  even  when  the 
reaction  of  the  potato  is  acid.  In  the  incubator  at  a 
temperature  of  37°  C.  a  transparent,  slightly  brownish 
or  yellowish-brown  growth,  somewhat  resembling  the 
growth  of  glanders,  is  produced.  It  contains  large 
numbers  of  long  spirals. 

In  bouillon  and  in  peptone  solution  the  cholera  organ- 
isms grow  well,  especially  upon  the  surface,  where  a 
folded,  wrinkled  mycoderma  is  formed.  Below  the  my- 
coderma  the  culture  fluid  generally  remains  clear.  If 
the  glass  be  shaken  and  the  mycoderma  broken  up, 
fragments  of  it  sink  to  the  bottom. 

In  milk  the  development  is  also  luxuriant,  but  takes 
place  in  such  a  manner  as  not  visibly  to  alter  its  appear- 


CHOLERA.  319 

ance.  The  existence  of  cholera  organisms  in  milk  is, 
however,  rather  short-lived,  for  the  occurrence  of  any 
acidity  at  once  destroys  them. 

Wolif hugel  and  Riedel  have  shown  that  if  the  spirilla 
are  planted  in  sterilized  water  they  grow  with  great  ra- 
pidity after  a  short  time,  and  can  be  found  alive  after 
months  have  passed.  Frankel  points  out  that  this  ability 
to  grow  and  remain  vital  for  long  periods  in  sterilized 
water  does  not  guarantee  the  same  power  in  unsterilized 
water,  for  in  the  latter  the  simultaneous  growth  of  other 
bacteria  in  a  few  days  serves  to  extinguish  the  cholera 
germs. 

One  of  the  characteristics  of  the  cholera  spirillum  is 
the  metabolic  production  of  indol.  The  detection  of  this 
substance  is  easy  if  the  spirilla  are  grown  in  a  transparent 
colorless  solution.  As  the  cholera  organisms  also  produce 
nitrites,  all  that  is  necessary  is  to  add  a  drop  or  two  of 
chemically  pure  sulphuric  acid  to  the  culture-medium 
for  the  production  of  the  well-known  reddish  color. 

Several  toxic  products  of  the  metabolism  of  the  spirilla 
have  been  isolated.  Brieger,  Frankel,  Roux  and  Yersin 
have  isolated  toxalbumins;  Villiers,  a  toxic  alkaloid  fatal 
to  guinea-pigs;  and  Gamaleia,  two  substances  about 
equally  toxic. 

The  cholera  spirilla  can  be  found  with  great  constancy 
in  the  intestinal  evacuations  of  all  cholera  cases,  and  can 
often  be  found  in  the  drinking-water,  milk,  and  upon 
vegetables,  etc.  in  cholera-infected  districts.  There  can 
be  little  doubt  that  they  find  their  way  into  the  body 
through  the  food  and  drink.  Many  cases  are  reported 
in  the  literature  upon  cholera  that  show  how  the  disease- 
germs  enter  the  drinking-water,  and  are  thus  distributed  ; 
how  they  are  sometimes  thoughtlessly  sprinkled  over  veg- 
etables, offered  for  sale  in  the  streets,  with  water  from 
polluted  gutters ;  how  they  enter  milk  with  water  used 
to  dilute  it ;  how  they  are  carried  about  in  clothing  and 
upon  foodstuffs ;  how  they  can  be  brought  to  articles  of 
food  upon  the  table  by  flies  which  have  preyed  upon 


320  PATHOGENIC  BACTERIA. 

cholera  excrement;  and  how  many  other  interesting  in- 
fections are  made  possible.  The  literature  upon  these 
subjects  is  so  vast  that  in  a  sketch  of  this  kind  it  is 
scarcely  possible  to  mention  even  the  most  instructive 
examples.  One  physician  is  reported  to  have  been  in- 
fected with  cholera  while  experimenting  with  the  spirilla 
in  Koch's  laboratory. 

The  evidence  of  the  specificity  of  the  cholera  spirillum 
when  collected  shows  that  it  is  present  in  the  choleraic 
dejections  with  great  regularity,  and  that  it  is  as  con- 
stantly absent  from  the  dejecta  of  healthy  individuals 
and  those  suffering  from  other  diseases ;  but  these  facts 
do  not  admit  of  satisfactory  proof  by  experimentation 
upon  animals.  Animals  are  never  affected  by  any  dis- 
ease similar  to  cholera  during  the  epidemics,  nor  do  foods 
mixed  with  cholera  discharges  or  with  pure  cultures  of 
the  cholera  spirillum  affect  them.  This  being  true,  we 
are  prepared  to  receive  the  further  information  that  sub- 
cutaneous injections  of  the  spirilla  are  often  without 
serious  consequences,  though  cultures  differ  very  much 
in  this  respect,  some  always  causing  a  fatal  septicemia  in 
guinea-pigs,  others  being  as  constantly  harmless. 

Intraperitoneal  injection  of  the  virulent  cultures  pro- 
duces a  fatal  peritonitis  in  guinea-pigs. 

One  reason  that  animals  and  certain  men  are  immune 
to  the  disease  seems  to  be  found  in  the  distinct  acidity 
of  the  normal  gastric  juice,  and  the  destruction  of  the  spi- 
rilla by  it.  Supposing  that  this  might  be  the  case,  Nicati 
and  Rietsch,  Von  Ermengen  and  Koch,  have  suggested 
methods  by  which  the  micro-organisms  can  be  introduced 
directly  into  the  intestine.  The  first-named  investigators 
ligated  the  common  bile-duct  of  guinea-pigs,  and  then  in- 
jected the  spirilla  into  the  duodenum  with  a  hypodermic 
needle.  The  result  was  that  the  animals  usually  died,  some- 
tiiiR-s  with  choleraic  symptoms  ;  but  the  excessively  grave 
nature  of  the  operation  upon  such  a  small  and  delicately 
constituted  animal  as  a  guinea-pig  greatly  lessens  the  value 
of  the  experiment.  Koch's  method  is  much  more  satisfac- 


CHOLERA.  321 

tory.  By  injecting  laudanum  into  the  abdominal  cavity 
of  guinea-pigs  the  peristaltic  movements  are  checked. 
The  amount  given  for  the  purpose  is  very  large,  about 
i  gram  for  each  200  grams  of  body-weight.  It  generally 
narcotizes  the  animals  for  a  short  time,  but  they  recover 
without  injury.  After  administering  the  opium  the  con- 
tents of  the  stomach  are  neutralized  by  introducing 
through  a  pharyngeal  catheter  5  c.cm.  of  a  5  per  cent, 
aqueous  solution  of  sodium  carbonate.  With  the  gastric 
contents  thus  alkalinized  and  the  peristalsis  paralyzed  a 
bouillon  culture  of  the  spirilla  is  introduced.  The  ani- 
mal recovers  from  the  manipulation,  but  shows  an  indis- 
position to  eat,  is  soon  observed  to  be  weak  in  the  pos- 
terior extremities,  subsequently  is  paralyzed,  and  dies 
within  forty-eight  hours.  The  autopsy  shows  the  intes- 
tine congested  and  filled  with  a  watery  fluid  rich  in  spi- 
rilla— an  appearance  which  Frankel  declares  to  be  exactly 
that  of  cholera.  In  man,  as  well  as  in  these  artificially 
injected  animals,  the  spirilla  are  never  found  in  the  blood 
or  the  tissues,  but  only  in  the  intestine,  where  they  fre- 
quently enter  between  the  basement  membrane  and  the 
epithelial  cells,  and  aid  in  the  detachment  of  the  latter. 

Issaeff  and  Kolle  found  that  when  virulent  cholera 
spirilla  are  injected  into  the  ear-veins  of  young  rabbits 
the  animals  die  on  the  following  day  with  symptoms  re- 
sembling the  algid  stage  of  human  cholera.  The  autopsy 
in  these  cases  showed  local  lesions  of  the  small  intestine 
very  similar  to  those  observed  in  cholera  in  man. 

Guinea-pigs  are  also  susceptible  to  intraperitoneal  in- 
jections of  the  spirillum,  and  speedily  succumb.  The 
symptoms  are — rapid  fall  of  temperature,  tenderness  over 
the  abdomen,  and  collapse.  The  autopsy  shows  an 
abundant  fluid  exudate  containing  the  micro-organism, 
and  injection  and  redness  of  the  peritoneum  and  viscera. 

Although  in  reading  upon  cholera  at  the  present  time 

we   find   very   little   skepticism    in   relation    to    Koch's 

"  comma  bacillus,"  we  do  find  occasional  doubters  who 

believe  with  Von  PettenkofFer  that  the  disease  is  mias- 

21 


32  2  PA  THOGENIC  BA  CTERIA. 

matic.  Pettenkoffer's  theory  is  that  the  disease  has 
much  to  do  with  the  ground-water  and  its  drying  zone. 
He  regards  as  the  principal  cause  of  the  disease  the  de- 
velopment of  germs  in  the  subsoil  moisture  during  the 
warm  months,  and  their  impregnation  of  the  atmosphere 
as  a  miasm  to  be  inhaled,  instead  of  ingested  with  food 
and  drink.  This  idea  of  Pettenkoffer's,  combined  with 
his  other  idea  that  individual  predisposition  must  pre- 
cede the  inception  of  the  disease,  is  scarcely  compatible 
with  what  has  gone  before,  and  cannot  possibly  be  made 
to  explain  the  march  of  the  disease  from  place  to  place 
with  caravans,  or  its  distribution  over  extended  areas 
when  fairs  and  religious  gatherings  among  the  Hindoos 
break  up,  the  people  from  an  infected  centre  carrying 
cholera  with  them  to  their  homes. 

While  it  is  an  organism  that  multiplies  with  great 
rapidity  under  proper  conditions,  the  cholera  spirillum 
is  not  possessed  of  much  resisting  power.  Sternberg 
found  that  it  was  killed  by  exposure  to  a  temperature 
of  52°  C.  for  four  minutes.  Kitasato,  however,  found 
that  ten  or  fifteen  minutes'  exposure  to  a  temperature 
°f  55°  C.  was  not  always  fatal.  In  the  moist  con- 
dition the  organism  may  retain  its  vitality  for  months, 
but  it  is  very  quickly  destroyed  by  desiccation,  as  was 
found  by  Koch,  who  observed  that  when  dried  in  a  thin 
film  its  power  to  grow  was  destroyed  in  a  few  hours. 
Kitasato  found  that  upon  silk  threads  the  vitality  might 
be  retained  longer.  Abel  and  Claussen  have  shown  that 
it  does  not  live  longer  than  twenty  to  thirty  days  in  fecal 
matter,  and  often  disappears  in  one  to  three  days.  The 
organism  is  very  susceptible  to  the  influence  of  carbolic 
acid,  bichlorid  of  mercury,  and  other  germicides. 

The  organism  is  also  destroyed  by  acids.  Hashimoto 
found  that  it  could  not  live  longer  than  fifteen  minutes 
in  vinegar  containing  2.2-3.2  per  cent,  of  acetic  acid. 

This  low  vital  resistance  of  the  microbe  is  very  fortu- 
nate, for  it  enables  us  to  establish  safeguards  for  the  pre- 
vention of  the  spread  of  the  disease.  Excreta,  soiled 


CHOLERA.  323 

clothing,  etc.  are  readily  rendered  harmless  by  the  proper 
use  of  disinfectants.  Water  and  foods  are  rendered  in- 
nocuous by  boiling  or  cooking.  Vessels  may  be  disin- 
fected by  thorough  washings  with  jets  of  boiling  water 
thrown  upon  them  through  hose.  Baggage  can  be  steril- 
ized by  superheated  steam. 

It  often  becomes  a  matter  of  importance  to  detect  the 
presence  of  cholera  in  drinking-water,  and,  as  the  dilu- 
tion in  which  the  bacteria  exist  in  such  a  liquid  may  be 
very  great,  much  difficulty  is  experienced  in  finding  them 
by  ordinary  methods.  One  of  the  most  expeditious  meth- 
ods that  have  been  recommended  is  that  of  Loffler,  who 
adds  200  c.cm.  of  the  water  to  be  examined  to  10  c.cm. 
of  bouillon,  allows  the  mixture  to  stand  in  an  incubator 
for  twelve  to  twenty-four  hours,  and  then  makes  plate- 
cultures  from  the  superficial  layer  of  the  liquid,  where, 
if  present,  the  development  of  the  spirilla  will  be  most 
rapid  because  of  the  presence  of  air.  A  similar  method 
can  be  used  to  detect  the  spirilla  when  their  presence  is 
suspected  in  feces. 

Gruber  and  Wiener,  Haffkine,  Pawlowsky,  and  Pfeiffer 
have  all  succeeded  in  immunizing  animals  against  the 
toxic  substances  removed  from  cholera  cultures  or  against 
living  cultures  properly  injected.  There  seems,  accord- 
ing to  the  researches  of  Pfeiffer,  to  be  no  doubt  that  in 
the  blood  of  the  protected  animals  a  protective  substance 
is  present.  In  the  peritoneal  infection  of  guinea-pigs 
the  spirilla  grow  vigorously  in  the  peritoneal  cavity,  and 
can  be  found  in  immense  numbers  after  twelve  to  twenty- 
four  hours.  If,  however,  together  with  the  culture  used 
for  inoculation,  a  few  drops  of  the  protective  serum  be  in- 
troduced, Pfeiffer  found  that  instead  of  multiplying  the 
organisms  underwent  a  peculiar  granular  degeneration 
and  disappeared,  the  unprotected  animal  dying,  the  pro- 
tected animal  remaining  well. 

Pfeiffer  and  Vogedes l  have  suggested  the  application 
of  this  "immunity-reaction"  for  the  positive  differentia- 

1  Centralbl.  fur  Bakt.  und  Parasitenk.,  March  21,  1896,  Bd.  xix.,  No.  n. 


324  PATHOGENIC  BACTERIA. 

tion  of  cholera  spirilla  in  cultures.  A  hanging-drop  of 
a  i  :  50  mixture  of  powerful  anti-cholera  serum  and  a 
particle  of  cholera  culture  is  made  and  examined  under 
the  microscope.  The  cholera  spirilla  at  once  become  in- 
active, and  are  in  a  short  time  converted  into  little  rolled- 
up  masses.  If  the  culture  added  be  a  spirillum  other 
than  the  true  spirillum  of  cholera,  instead  of  destruc- 
tion of  the  micro-organisms  following  exposure  to  the 
serum,  they  multiply  and  thrive  in  the  mixture  of  serum 
and  bouillon. 

The  specific  immunity-reaction  of  the  cholera  serum 
has  been  carefully  studied  by  Loburnheim,1  and  is 
specific  against  cholera  alone.  The  protection  is  not 
due  to  the  strongly  bactericidal  property  of  the  serum, 
but  to  its  stimulating  effect  upon  the  body-cells.  If 
the  serum  be  heated  to  6o°-7O°  C.,  and  its  bactericidal 
power  thus  destroyed,  it  is  still  capable  of  producing 
immunity. 

The  immunity  produced  by  the  injection  of  the  spirilla 
into  guinea-pigs  continues  in  some  cases  as  long  as  four 
and  a  half  months,  but  the  power  of  the  serum  to  con- 
fer immunity  is  lost  much  sooner. 

Of  the  numerous  attempts  which  have  from  time  to 
time  been  made,  and  are  still  being  made,  to  produce 
immunity  against  cholera  in  man  or  to  cure  cholera 
when  once  established  in  the  human  organism,  nothing 
very  favorable  can  at  the  present  time  be  said.  Experi- 
ments in  this  field  are  not  new :  we  find  Dr.  Ferran  ad- 
ministering hypodermic  injections  of  pure  virulent  cul- 
tures of  the  cholera  spirillum  in  Spain  as  early  as  1885, 
in  the  hope  of  bringing  about  immunity.  The  more  mod- 
ern work  of  Haffkine  seems  to  be  followed  by  a  distinct 
diminution  of  mortality  in  protected  individuals.  Ac- 
cording to  the  work  of  this  investigator,  two  vaccines  are 
used,  one  of  which,  being  mild,  prepares  the  animal  (or 
man)  for  a  powerful  vaccine,  which,  were  it  not  preceded 
by  the  weaker  form,  would  bring  about  extensive  tissue- 

1  Zeitschrift  fiir  Hygiene,  xx.,  p.  438. 


CHOLERA,  325 

necrosis  and  perhaps  death.  Protection  certainly  seems 
to  follow  the  operation  of  these  vaccines. 

Haffki lie's  studies  embrace  more  than  40,000  inocula- 
tions performed  in  India.  From  his  latest  paper  (Dec., 
1895)  the  following  extract  will  show  the  results : 

ui.  In  all  those  instances  where  cholera  has  made  a 
large  number  of  victims,  that  is  to  say,  where  it  has 
spread  sufficiently  to  make  it  probable  that  the  whole 
population,  inoculated  and  uninoculated,  were  equally 
exposed  to  the  infection, — in  all  these  places  the  results 
appeared  favorable  to  inoculation. 

"2.  The  treatment  applied  after  an  epidemic  actually 
breaks  out  tends  to  reduce  the  mortality  even  during  the 
time  which  is  claimed  for  producing  the  full  effect  of  the 
operation.  In  the  Goya  Garl,  where  weak  doses  of  a 
relatively  weak  vaccine  had  been  applied,  this  reduction 
was  to  half  the  number  of  deaths  ;  in  the  coolies  of  the 
Assam-Burmah  survey-party,  where,  as  far  as  I  can  gather 
from  my  preliminary  information,  strong  doses  have  been 
applied,  the  number  of  deaths  was  reduced  to  one-seventh. 
This  fact  would  justify  the  application  of  the  method  in- 
dependently of  the  question  as  to  the  exact  length  of  time 
during  which  the  effect  of  this  vaccination  lasts. 

"3.  In  Lucknow,  where  the  experiment  was  made  on 
small  doses  of  weak  vaccines,  a  difference  in  cases  and 
deaths  was  still  noticeable  in  favor  of  the  inoculated 
fourteen  to  fifteen  months  after  vaccination  in  an  epidemic 
of  exceptional  virulence.  This  makes  it  probable  that  a 
protective  effect  could  be  obtained  even  for  long  periods 
of  time  if  larger  doses  of  a  stronger  vaccine  were  used. 

"4.  The  best  results  seem  to  be  obtained  from  applica- 
tion of  middle  doses  of  both  anticholera  vaccines,  the 
second  one  being  kept  at  the  highest  possible  degree  of 
virulence  obtainable. 

"  5.  The  most  prolonged  observations  on  the  effect  of 
middle  doses  were  made  in  Calcutta,  where  the  mortality 
from  the  eleventh  up  to  the  four  hundred  and  fifty-ninth 
day  after  vaccination  was,  among  the  inoculated,  17.24 


326  PA  THOGENIC  BA  CTERIA . 

times  smaller,  and  the  number  of  cases  19.27  times 
smaller  than  among  the  not  inoculated." 

Pawlowsky  and  others  have  found  that  the  dog  is  sus- 
ceptible to  cholera,  and  have  utilized  the  observation  to 
prepare  an  antitoxic  serum  in  considerable  quantities. 
The  dogs  were  first  immunized  with  attenuated  cultures, 
then  with  more  and  more  virulent  cultures,  until  a  serum 
was  obtained  whose  value  was  estimated  at  i  :  130,000 
upon  experimental  animals. 

Freymuth  and  others  have  endeavored  to  secure  favor- 
able results  from  the  injection  of  blood-serum  from  con- 
valescent patients  into  the  diseased.  One  recovery  out 
of  three  cases  treated  is  recorded — not  a  very  glittering 
result. 

In  all  these  preliminaries  the  foreshadowing  of  a  future 
therapeusis  must  be  evident,  but  as  yet  nothing  really 
satisfactory  has  been  achieved. 

SPIRILLA  RESEMBLING  THE  CHOLERA  SPIRILLUM. 

The  Finkler  and  Prior  Spirillum. — Somewhat  similar 
to  the  spirillum  of  cholera,  and  in  some  respects  closely 
related  to  it,  is  the  spirillum  obtained  from  the  feces  of 
a  case  of  cholera  nostras  by  Finkler  and  Prior  in  1884. 
It  is  a  rather  shorter,  stouter  organism,  with  a  more  pro- 
nounced curve,  than  the  cholera  spirillum,  and  rarely 
forms  the  long  spirals  which  characterize  the  latter. 
The  central  portion  is  also  somewhat  thinner  than  the 
ends,  which  are  a  little  pointed  and  give  the  organism 
a  less  uniform  appearance  than  that  of  cholera  (Fig.  84). 
Involution-forms  are  very  common  in  cultures,  and  occur 
as  spheres,  spindles,  clubs,  etc.  Like  the  cholera  spiril- 
lum, each  organism  is  provided  with  a  single  flagellum 
situated  at  its  end,  and  is  actively  motile.  Although  at 
first  thought  to  be  a  variety  of  the  cholera  germ,  marked 
differences  of  growth  were  soon  observed,  and  showed 
the  organism  to  be  a  separate  species. 

The  growth  upon  gelatin  plates  is  quite  rapid,  and  leads 
to  such  extensive  liquefaction  that  four  or  five  dilutions 


SPIRILLA   RESEMBLING   CHOLERA.  327 

must  frequently  be  made  before  the  growth  of  a  single 
colony  can  be  observed.     To  the  naked  eye  the  colqnies 


x_    ^       «>    ^^    J9*      «k*»  *• 

"W4c§gg§? 

-^i^_.     iNN«, T«miV 


FIG.  84. — Spirillum  of  Finkler  and  Prior,  from  an  agar-agar  culture ;    x  1000 
(Itzerott  and  Niemann). 

appear  as  small  white  points  in  the  depths  of  the  gelatin 
(Fig.   85).     They,   however,    rapidly  reach  the   surface, 


FIG.  85. — Spirillum  of  Finkler  and  Prior:   colony  twenty-four  hours   old,  as 
seen  upon  a  gelatin  plate;    x   100  (Frankel  and  Pfeiffer). 

begin   liquefaction   of  the   gelatin,    and  by  the   second 


328 


PATHOGENIC  BACTERIA. 


day  appeaf  about  the  size  of  lentils,  and  are  situated  in 
little  depressions.  Under  the  microscope  they  are  of  a 
yellowish-brown  color,  are  finely  granular,  and  are  sur- 
rounded by  a  zone  of  sharply  circumscribed  liquefied 
gelatin.  Careful  examination  with  a  high  power  of  the 
microscope  shows  a  rapid  movement  of  the  granules  of 
the  colony. 

In  gelatin  punctures  the  growth  takes  place  rapidly 
along  the  whole  puncture,  forming  a  stocking-shaped 
liquefaction  filled  with  cloudy  fluid  which  does  not  pre- 
cipitate rapidly  ;  a  rather  smeary,  whitish  mycoderma  is 
generally  formed  upon  the  surface.  The  much  more  ex- 
tensive and  more  rapid  liquefaction  of  the  medium,  the 
wider  top  to  the  funnel-shaped  liquefaction  at  the  surface, 


FIG.   86. — Spirillum   of  Finkler  and  Prior :   gelatin  puncture-cultures   aged 
forty -eight  and  sixty  hours  (Shakespeare). 

the  absence  of  the  air-bubble,  and  the  clouded  nature  of 
the  liquefied  material,  all  serve  to  differentiate  it  from  the 
cholera  spirillum. 

Upon  agar-agar  the  growth  is  also  very  rapid,  and  in 
a  short  time  the  whole  surface  of  the  culture-medium  is 


SPIRILLA  RESEMBLING   CHOLERA.  329 

covered  with  a  moist,  thick,  slimy  coating,  which  may 
have  a  slightly  yellowish  tinge. 

The  cultures  upon  potato  are  also  very  different  from 
those  of  cholera,  for  instead  of  a  temperature  of  37°  C. 
being  required  for  a  rapid  development,  the  Finkler  and 
Prior  spirilla  grow  rapidly  at  the  room-temperature,  and 
produce  a  grayish-yellow,  slimy,  shining  layer,  which 
may  cover  the  whole  of  the  culture-medium. 

Blood-serum  is  rapidly  liquefied  by  the  growth  of  the 
organism. 

Buchner  has  shown  that  in  media  containing  some 
glucose  an  acid  reaction  is  produced. 

The  spirillum  does  not  grow  well,  if  at  all,  in  milk, 
and  speedily  dies  in  water. 

The  organism  does  not  produce  indol. 

The  spirillum  can  be  stained  well  by  the  ordinary 
dyes,  and  seems,  like  the  cholera  spirillum,  to  have  a 
special  affinity  for  the  aqueous  solution  of  fuchsin. 

In  connection  with  this  bacillus  the  question  of  patho- 
genesis  is  a  very  important  one.  At  first  it  was  sus- 
pected that  it  was,  if  not  the  spirillum  of  cholera  itself, 
a  very  closely  allied  organism.  Later  it  was  regarded 
as  the  cause  of  cholera  nostras.  At  present  its  exact 
pathological  significance  is  a  question.  It  was  in  one 
case  secured  by  Knisl  from  the  feces  of  a  suicide,  and 
has  been  found  in  carious  teeth  by  Miiller. 

When  injected  into  the  stomach  of  guinea-pigs  treated 
according  the  method  of  Koch,  about  30  per  cent,  of  the 
animals  die,  but  the  intestinal  lesions  produced  are  not 
the  same  as  those  produced  by  the  cholera  spirillum. 
The  intestines  in  such  cases  are  pale  and  filled  with 
watery  material  having  a  strong  putrefactive  odor.  This 
fluid  teems  with  the  spirilla. 

It  seems  very  unlikely,  from  the  collected  evidence, 
that  the  Finkler  and  Prior  spirillum  is  associated  with 
pathogenesis  in  the  human  species.  As  Frankel  points 
out,  it  is  probably  a  frequent  and  harmless  inhabitant  of 
the  human  intestine. 


330  PATHOGENIC  BACTERIA. 

The  Spirillum  of  Denecke. — Another  organism  with 
a  distinct  resemblance  to  the  cholera  spirillum  is  one 
described  by  Denecke  as  occurring  in  old  cheese  (Fig. 
87).  Its  form  is  much  the  same  as  that  of  the  spirillum 
of  cholera,  the  shorter  individuals  being  of  equal  diameter 
throughout.  The  spirals  which  are  produced  are  longer 
than  those  of  the  Finkler  and  Prior  spirillum,  and  are 
more  tightly  coiled  than  those  of  the  cholera  spirillum. 

Like  its  related  species,  this  micro-organism  is  actively 
motile.  It  grows  at  the  room-temperature,  as  well  as  at 
37°  C.,  in  this  respect,  as  in  its  reaction  to  stains,  much 
resembling  the  other  two. 

Upon  gelatin  plates  the  growth  of  the  colonies  is  much 
more  rapid  than  that  of  the  cholera  spirillum,  but  slower 
than  that  of  the  Finkler  and  Prior  •spirillum.  The  col- 


FIG.  87. — Spirillum  Denecke,  from  an  agar-agar  culture ;    x  1000  (Itzerott 
and  Niemann). 

onies  appear  as  small  whitish,  round  points,  which  soon 
reach  the  surface  of  the  gelatin  and  commence  liquefac- 
tion. By  the  second  day  they  are  about  the  size  of  a 
pin's  head,  have  a  yellow  color,  and  occupy  the  bottom 
of  a  conical  depression.  The  appearance  is  much  like 
that  of  a  plate  of  cholera  spirilla. 

The  microscope  shows  the  colonies  to  be  of  irregular 


SPIRILLA   RESEMBLING   CHOLERA.  331 

shape  and  coarsely  granular.  The  color  is  yellow,  and  is 
pale  at  the  edges,  gradually  becoming  intense  toward  the 
centre.  The  colonies  are  surrounded  at  first  by  distinct 
lines  of  circumscription,  later  by  clear  zones,  which,  ac- 
cording to  the  illumination,  are  pale  or  dark.  From  this 
description  it  will  be  seen  that  the  colonies  differ  from 
those  of  cholera  in  the  prompt  liquefaction  of  the  gelatin, 
their  rapid  growth,  yellow  color,  irregular  form,  and  dis- 
tinct lines  of  circumscription. 

In  gelatin  punctures  the  growth  takes  place  all  along 
the  track  of  the  wire,  and  forms  a  cloudy  liquid  which 
precipitates  at  the  apex  in  the  form  of  a  coiled  mass. 
Upon  the  surface  a  delicate  imperfect  yellowish  myco- 


FlG.  88. — Spirillum  Denecke  :    gelatin    puncture- cultures  aged  forty-eight  and 
sixty  hours  (Shakespeare). 

derma  forms.  Liquefaction  of  the  entire  gelatin  gen- 
erally requires  about  two  weeks. 

Upon  agar-agar  this  spirillum  grows  as  a  thin  yellow- 
ish layer  which  does  not  seem  inclined  to  spread  widely. 

The  culture  upon  potato  is  luxuriant  if  grown  in  the 
incubating  oven.  It  appears  as  a  distinct  yellowish  moist 


332  PATHOGENIC  BACTERIA. 

film,  and  when  examined  microscopically  is  seen  to  con- 
tain long  beautiful  spirals. 

The  organism  sometimes  produces  indol,  but  is  irreg- 
ular in  its  action  in  this  respect. 

The  spirillum  of  Denecke  is  mentioned  only  because 
of  its  morphological  relation  to  the  cholera  spirillum, 
not  because  of  any  pathogenesis  which  it  possesses.  It 
probably  is  not  associated  with  any  human  disease.  Ex- 
periments, however,  have  shown  that  when  the  spirilla 
are  introduced  into  the  intestines  of  guinea-pigs  whose 
gastric  contents  are  alkalinized  and  whose  peristalsis  is 


v  '  >  v*-     ^*-  -  ,  .    ir .-. 

x%<-r*iUHi  £# 

r.\  >V  ',*>»y  s 
V-*v-%if^       /    ^' - 

^  . %  *T^^    •      rf       *^ 


FIG.   89.— Spirillum   Metchnikoff,  from  an  agar-agar  culture ;    x  1000  (Itzerott 
and  Niemann). 

paralyzed  with  opium,  about  20  per  cent,  of  the  animals 
die  from  intestinal  disease. 

The  Spirillum  of  Gamale'ia  (Spirillum  Metchnikoff). 
-Very  closely  related  to  the  cholera  spirillum  in  its 
morphology  and  vegetation  and  possibly,  as  has  been 
suggested,  a  descendant  of  the  same  original  stock,  is  the 
spirillum  which  Gamale'ia  cultivated  from  the  intestines 
of  chickens  affected  with  a  disease  similar  to  chicken- 
cholera.  This  spirillum  is  a  curved  organism,  a  trifle 
shorter  and  thicker  than  the  cholera  spirillum,  a  little 
more  curved,  and  with  similar  rounded  ends  (Fig.  89). 


SPIRILLA   RESEMBLING   CHOLERA.  333 

It  forms  long  spirals  in  appropriate  media,  and  is  actively 
motile.  Bach  spirillum  is  provided  with  a  terminal  flagel- 
lum.  No  spores  have  been  positively  demonstrated. 

The  organism,  like  the  cholera  vibrio,  is  very  suscep- 
tible to  the  influence  of  acids,  high  temperatures,  and 
drying,  so  that  spores  are  probably  not  formed.  It  grows 
well  both  at  the  temperature  of  the  room  and  at  that  of 
incubation. 

The  thermal  death-point  is  50°  C.,  continued  for  five 
minutes. 

The  bacterium  stains  easily,  the  ends  more  deeply  than 
the  center.  It  is  not  stained  by  Gram's  method. 

Upon  gelatin  plates   a   remarkable   similarity  to   the 


FIG.  90. — Spirillum  Metschnikoff;  puncture-culture  in  gelatin  forty-eight  hours 
old  (Frankel  and  Pfeiffer). 

colonies  of  the  cholera  spirillum  is  developed,  yet  there 
is  a  difference,  and  Pfeiffer  points  out  that  "it  is  com- 
paratively easy  to  differentiate  between  a  plate  of  pure 
cholera  spirillum  and  a  plate  of  pure  Spirillum  Metch- 
nikoff,  yet  it  is  almost  impossible  to  pick  out  a  few 
colonies  of  the  latter  if  mixed  upon  a  plate  with  the 
former. ' ' 

Frankel  regards  this  bacterium  as  a  kind  of  interme- 


334  PATHOGENIC  BACTERIA, 

diate  species  between  the  cholera  and  the  Finkler-Prior 
spirilla. 

The  colonies  upon  gelatin  plates  appear  in  about  twelve 
hours  as  small  whitish  points,  and  rapidly  develop,  so  that 
by  the  end  of  the  third  day  large  saucer-shaped  areas  of 
liquefaction  resembling  colonies  of  the  Finkler-Prior 
spirilla  occur.  The  liquefaction  of  the  gelatin  is  quite 
rapid,  the  resulting  fluid  being  turbid.  Generally  there 
will  be  upon  a  plate  of  Vibrio  Metchnikoff  some  colo- 
nies which  closely  resemble  cholera  by  occupying  small 
conical  depressions  in  the  gelatin.  Under  a  high  power 
of  the  microscope  the  contents  of  the  colonies,  which  ap- 
pear to  be  of  a  brownish  color,  are  observed  to  be  in  rapid 
motion.  The  edges  of  the  bacterial  mass  are  fringed  with 
radiating  organisms  (Fig.  90). 

In  gelatin  tubes  the  culture  is  very  much  like  that  of 
cholera,  but  develops  more  slowly. 

Upon  the  surface  of  agar-agar  a  yellowish-brown 
growth  develops  along  the  whole  line  of  inoculation. 

On  potato  at  the  room-temperature  no  growth  occurs, 
but  at  the  temperature  of  the  incubator  a  luxuriant 
yellowish-brown  growth  takes  place.  Sometimes  the 
color  is  quite  dark,  and  chocolate-colored  potato  cultures 
are  not  uncommon. 

In  bouillon  the  growth  which  occurs  at  the  tempera- 
ture of  the  incubator  is  quite  characteristic,  and  very 
different  from  that  of  the  cholera  spirillum.  The  entire 
medium  becomes  clouded,  of  a  grayish-white  color,  and 
opaque.  A  folded  and  wrinkled  mycoderma  forms  upon 
the  surface. 

When  glucose  is  added  to  the  bouillon  no  fermentation 
or  gas-production  results. 

When  grown  in  litmus  milk  the  original  blue  color  is 
changed  to  pink  in  a  day,  and  at  the  end  of  another  day 
the  color  is  all  destroyed  and  the  milk  coagulated.  Ulti- 
mately the  clots  of  casein  sediment  in  irregular  masses, 
and  clear  colorless  whey  is  supernatant. 

The  addition  of  sulphuric  acid  to  a  culture  grown  in  a 


SPIRILLA   RESEMBLING   CHOLERA.  335 

medium  rich  in  peptone  produces  the  same  rose  color 
observed  in  cholera  cultivations. 

The  organism  is  pathogenic  for  animals,  but  not  for 
man.  PfeifFer  has  shown  that  chickens,  pigeons,  and 
guinea-pigs  are  highly  susceptible  animals.  The  birds 
when  inoculated  under  the  skin  generally  die — pigeons 
always.  W.  Rindfleish  has  pointed  out  that  this  positive 
fatal  outcome  of  the  introduction  of  the  spirillum  into 
pigeons  makes  it  a  valuable  diagnostic  point  for  the 
differentiation  of  this  spirillum  from  that  of  cholera. 
According  to  his  researches,  the  simple  subcutaneous  in- 
jection of  the  most  virulent  cholera  cultures  is  never 
fatal  to  pigeons.  The  birds  only  die  when  the  injections 
are  made  into  the  muscles  in  such  a  manner  that  the 
muscular  tissue  is  injured  and  becomes  a  locus  minoris 
resistentice.  When  guinea-pigs  are  treated  according  to 
the  method  of  Koch  for  the  inoculation  of  cholera,  the 
temperature  of  the  animal  rises  for  a  short  time,  then 
abruptly  falls  to  33°  C.  or  less.  Death  follows  in  twenty 
to  twenty-four  hours.  A  distinct  inflammation  of  the 
intestine,  with  exudate  and  numerous  spirilla,  may  be 
found.  The  spirilla  can  also  be  found  in  the  heart's 
blood  and  in  the  organs  of  such  guinea-pigs.  When  the 
bacilli  are  introduced  by  subcutaneous  inoculation,  the 
autopsy  shows  a  bloody  edema  and  a  superficial  necrosis 
of  the  tissues. 

In  the  blood  and  all  the  organs  of  pigeons  and  young 
chickens  the  organisms  can  be  found  in  such  large  num- 
bers that  Pfeiffer  has  suggested  the  term  "  vibrionensep- 
ticsemie"  for  the  condition.  In  the  intestines  very  few 
alterations  are  noticeable,  and  very  few  spirilla  can  be 
found. 

Gamaleia  has  shown  that  pigeons  and  guinea-pigs  can 
be  made  immune  by  inoculating  them  with  cultures  ster- 
ilized for  a  time  at  a  temperature  of  100°  C.  Mice  and 
rabbits  are  immune  except  to  very  large  doses. 

Spirillum  Berolinensis. — This  organism  (Fig.  91), 
which  was  discovered  by  Neisser  in  the  summer  of  1893, 


PATHOGENIC  BACTERIA. 

is  of  great  interest  in  comparison  with  the  spirillum  of 
cholera  and  its  related  forms.  Its  morphology  is  in  every 
particular  exactly  like  that  of  the  cholera  spirillum,  but 
its  growth  is  a  little  more  rapid.  It  grows  upon  the 
same  culture-media  and  at  the  same  temperature.  The 
colonies  are,  however,  quite  different. 

Upon  the  second  day,  when  grown  upon  gelatin 
plates,  the  colonies  of  the  Spirillum  Berolinensis  appear 
finely  granular  and  paler  than  those  of  cholera.  The 
borders  are  generally  smooth  and  circular.  As  it  be- 
comes older  the  colony  takes  on  a  slightly  brownish 
color,  and  may  be  nodulated  or  radiately  lobulated.  The 
gelatin  is  very  slowly  liquefied. 


'•V 


FIG.  91. — Spirillum  Berolinensis,  from  an  agar-agar  culture;  x  1000  (Itzerott 
and  Niemann). 

In  puncture-cultures  the  development  takes  place  along 
the  entire  puncture,  and  causes  a  gradual  liquefaction  of 
the  gelatin. 

Upon  agar-agar  the  growth  is  generally  similar  to  that 
of  the  cholera  spirillum,  but  at  times  is  copious,  dry, 
and  ragged,  and  suggests  leather  by  its  appearance. 

When  introduced  intraperitoneally  into  guinea-pigs 
the  animals  die  in  from  one  to  two  days. 

The  indol  reaction  is  exactly  like  that  given  by  cul- 


SPIRILLA   RESEMBLING   CHOLERA.  337 

tures  of  the  cholera  spirillum.     The  spirillum  does  not 
stain  by  Gram's  method. 

Spirillum  Dunbar.  —  This  organism  (Fig.  92)  was  de- 


V 


FIG.  92.  —  Spirillum  Dunbar,  from  agar-agar;   x   1000  (Itzerott  and  Niemann). 

scribed  in  1893  by  Dunbar  and  Oergel,  who  secured  it 
from  the  water  of  the  Elbe  River.  It  much  resembles 
the  cholera  spirillum,  but  it  never  exhibits  sigmoid  forms. 
It  stains  poorly,  the  ends  taking  the  color  much  better 
than  the  central  portion. 

Gelatin  is  liquefied  by  the  growth  of  this  organism 
more  quickly  than  by  the  cholera  spirillum.  The  colo- 
nies upon  gelatin  and  the  puncture-cultures  in  gelatin 
are  identical  with  those  of  the  cholera  spirillum. 

On  agar-agar  a  luxuriant  whitish-yellow  layer  is  pro- 
duced. 

In  bouillon  and  peptone  solution  the  addition  of  dilute 
sulphuric  acid  produces  the  red  color  of  nitro-indol. 

It  is  said  that  cultures  grown  at  a  temperature  of  22°  C. 
phosphoresce  in  the  dark. 

The  spirillum  seems  to  be  pathogenic  for  guinea-pigs 
when  introduced  into  the  stomach  according  to  Koch's 
method  for  cholera. 

Spirillum   Danubicus.  —  This  organism  (Fig.  93)  also 

22 


PATHOGENIC  BACTERIA, 

much  resembles  cholera.  It  was  first  isolated  by  Heider 
in  1892.  In  appearance  it  is  rather  delicate  and  decidedly 
curved.  It  is  often  united  in  sigmoid  and  semicircular 
forms,  and  exhibits  long  spirals  in  old  cultures.  It  is 
actively  motile,  each  organism  presenting  a  terminal 
flagellum. 

The  growth  upon  gelatin  plates  is  rapid.  Small  light- 
gray  colonies,  resembling  those  of  cholera,  but  exhibit- 
ing a  dentate  margin,  are  observed.  The  growth  in 
gelatin  punctures  also  much  resembles  cholera,  and  the 
agar-agar  growth  can  scarcely  be  distinguished  from  it. 

The  potato  growth  has  a  distinct  yellowish-brown 
color. 

Milk  is  coagulated  in  three  or  four  days. 


FIG.  93. — Spirillum  Danubicus,  from  an  agar-agar  culture;  x  1000  (Itzerott  and 

Niemann). 

This  spirillum  does  not  produce  indol. 

Heider  found  the  spirillum  pathogenic  for  guinea-pigs. 

Spirillum  I.  of  Wernicke. — This  organism  is  about 
twice  as  large  as  the  cholera  spirillum,  liquefies  gelatin 
more  rapidly,  produces  indol,  and  is  feebly  pathogenic 
for  guinea-pigs. 

Spirillum  II.  of  Wernicke. — This  spirillum  is  smaller 
than  the  cholera  spirillum,  liquefies  gelatin  more  slowly, 


SPIRILLA   RESEMBLING  CHOLERA.  339 

produces   indol,  and   is   highly   pathogenic   for  rabbits, 
guinea-pigs,  pigeons,  and  mice. 

Spirillum    Bonhoffi. — This    organism   (Fig.    94)   was 
found   in  water  by  Bonhoff.     It   has   a   decided   resem- 


y 

V-   ^ 
«V4 


«  \ 

FIG.  94. — Spirillum  Bonhoffi,  from  a  culture  upon  agar-agar;    x  1000  (Itzerott 
and  Niemann). 

blance  to  the  cholera  spirillum,  but  is  rather  stouter 
and  less  curved.  Curved  forms — i.  e.  semicircles,  sig- 
moids,  and  spirals — occur  in  old  cultures  especially. 

These  organisms  are  colored  badly  with  ordinary  stains, 
dahlia  seeming  to  be  the  most  appropriate  color,  and  ac- 
complishing the  process  better  if  warmed.  The  organ- 
ism is  motile,  and  has  a  long  flagellum  attached  to  one 
end. 

The  colonies  develop  slowly  upon  gelatin  plates,  first 
appearing  in  forty-eight  hours  as  little  grayish  points. 
The  margin  of  the  colony  is  sharply  circumscribed  ;  the 
interior  is  broken  up.  The  gelatin  is  not  liquefied.  In 
gelatin  punctures  there  is  no  liquefaction  observable. 

Upon  agar-agar  the  development  at  the  temperature 
of  the  incubator,  which  is  more  rapid  than  that  at  the 
temperature  of  the  room,  results  in  the  production  of  a 
bluish-gray  layer. 

The  growth  upon  potato  has  a  brownish  color.     The 


340  PA  THOGENIC  BA  CTERIA . 

growth  in  bouillon  and  in  peptone  solutions  is  accompa- 
nied by  the  production  of  indol. 

The  spirillum  is  pathogenic  for  mice,  guinea-pigs,  and 
canary  birds. 

Spirillum  Weibeli.— This  spirillum  (Fig.  95)  was  found 
in  1892  by  Weibel  in  spring-water  which  had  a  long  time 


FIG.  95.  —  Spirillum  Weibeli,  from  agar-agar;  x  icxx>  (Itzerott  and  Niemann). 


before  been  infected  by  cholera.  It  is  short,  rather  thick, 
and  distinctly  bent,  often  forming  S-shaped  figures. 

The  colonies  before  liquefaction  sets  in  are  described 
as  pale-brown,  transparent,  circular,  and  homogeneous. 
Liquefaction  is  much  more  rapid  than  in  cholera,  and 
causes  the  borders  of  the  colonies  to  become  irregular. 
In  the  centre  of  each  colony  a  little  depression  is  ob- 
served. 

In  gelatin  puncture-cultures  the  growth  is  rapid,  be- 
ginning first  upon  the  surface,  where  a  large  flat,  saucer- 
shaped  liquefaction,  extending  to  the  sides  of  the  tube, 
forms.  Scarcely  any  growth  takes  place  in  the  puncture, 
but  the  superficial  liquefaction,  separated  by  a  horizontal 
line  from  the  normal  gelatin,  descends  slowly. 

Upon  agar-agar  a  grayish-white  layer  is  formed. 

No  growth  has  been  obtained  upon  potato. 


SPIRILLA   RESEMBLING   CHOLERA.  341 

In  alkaline  peptone  solution  a  slow  but  luxuriant 
growth  takes  place. 

Spirillum  Milleri. — This  spirillum  (Fig.  96)  was  found 
in  the  mouth  by  Miller  in  1885.  It  resembles  the  cholera 


FIG.  96. — Spirillum  Milleri,  from  an  agar-agar  culture;    x  1000  (Itzerott  and 

Niemann). 

spirillum  somewhat,  but  is  much  more  like  the  spirillum 
of  Finkler  and  Prior,  with  which  many  bacteriologists 
think  it  identical. 

Upon  gelatin  the  colonies  are  small,  finely  granular, 
have  a  narrow  border-zone  and  a  pale-brown  color.  The 
gelatin  is  rapidly  liquefied. 

Upon  agar-agar  a  thick  yellowish  layer  is  produced. 

The  organism  seems  not  to  be  pathogenic. 

Spirillum  Aquatilis. — Giinther  in  1892  found  this  or- 
ganism (Fig.  97)  in  the  water  of  the  river  Spree.  It  is 
similar  to  the  cholera  spirillum  in  shape,  has  a  long 
terminal  flagellum,  and  is  motile. 

The  colonies  which  form  upon  gelatin  are  circular, 
have  smooth  borders,  and  look  very  much  as  if  bored  out 
with  a  tool.  They  have  a  brown  color  and  are  finely 
granular.  In  gelatin  puncture-cultures  the  growth  occurs 
almost  exclusively  at  the  surface. 

The  agar-agar  cultures  are  similar  to  those  of  cholera. 

Scarcely  any  development  occurs  in  bouillon.     By  the 


342  PATHOGENIC  BACTERIA. 

growth  of  the  organism  sulphuretted  hydrogen  gas  is 
produced. 

The  spirillum  does  not  grow  at  all  upon  potato. 

Giinther  did  not  find  the  organism  to  be  pathogenic. 

Spirillum  Terrigenus. — This  species,  also  discovered 
by  Giinther,  was  secured  from  earth.  It  generally  occurs 
in  a  slightly  curved  form,  but  sometimes  is  spiral.  It  is 
actively  motile  and  has  a  terminal  flagellum. 

The  colonies,  which  appear  in  twenty-four  hours,  are 
small,  structureless,  and  transparent,  and  later  take  on  a 
*  *  fat-drop ' '  appearance. 

Upon  agar-agar  a  thin  white  coating  is  formed.  Milk 
is  coagulated  by  the  growth  of  the  organism.  No  indol 
is  produced. 

The  organism  does  not  stain  by  Gram's  method,  and 
is  said  not  to  be  pathogenic  for  guinea-pigs  or  for  mice. 


.  tt»s   "\  \-3  - 


FlG.  97. — Spirillum  aquatilis,  from  an  agar-agar  culture ;    x  1000  (Itzerott  and 

Niemann). 

Vibrio  Schuylkiliensis.— This  form,  closely  resembling 
the  cholera  spirillum,  was  found  by  Abbott1  in  sewage- 
polluted  water  from  the  Schuylkill  River  at  Philadelphia. 
The  colonies  upon  gelatin  plates  resemble  very  closely 
those  of  Spirillum  Metschnikovi.  In  gelatin  puncture- 
cuHures  the  appearance  isexactly  like  the  true  cholera  spir- 

'  Jour,  of  Exper.  MeJ.t  vol.  i.,  No.  3,  July,  1896,  p.  419. 


SPIRILLA  RESEMBLING  CHOLERA.  343 

ilium.  At  times  the  growth  may  be  a  little  more  rapid. 
The  growth  on  agar  is  very  luxuriant,  and  gives  off  a 
pronounced  odor  of  indol.  Loffler's  blood-serum  is  ap- 
parently not  a  perfectly  adapted  medium,  but  upon  it  the 
organisms  grow,  with  resulting  liquefaction.  Upon  po- 
tato at  the  point  of  inoculation  there  is  a  thin,  glazed, 
more  or  less  dirty  yellow,  shading  to  brownish  deposit  that 
is  sometimes  surrounded  by  a  flat,  dry,  lusterless  zone. 

In  litmus  milk  a  slightly  reddish  tinge  is  found  after 
twenty-four  hours  at  body  temperature.  After  forty-eight 
hours  this  is  increased  and  the  milk  is  coagulated.  In 
peptone  solutions  indol  is  produced.  No  gas  is  pro- 
duced in  glucose-containing  culture-media.  The  organ- 
ism is  a  facultative  anaerobic  spirillum.  The  thermal 
death-point  is  50°  C.  for  five  minutes. 

The  organism  is  pathogenic  for  pigeons,  guinea-pigs, 
and  mice.  The  pathogenesis  is  much  like  that  of  the 
Spirillum  Metschnikovi.  No  PfeifFer's  phenomenon  was 
observed  with  the  use  of  the  serum  of  immunized  ani- 
mals. 

Immunity  was  produced  in  pigeons,  and  it  was  found 
that  their  serum  was  protective  against  both  the  Vibrio 
Schuylkiliensis  and  Spirillum  Metschnikovi,  the  immun- 
ity thus  produced  being  of  about  ten  days'  duration. 

In  a  second  paper  by  Abbott  and  Bergy l  it  was  shown 
that  the  vibrios  were  found  in  river  water  during  all 
four  seasons  of  the  year,  and  in  all  parts  of  the  river 
within  the  city,  both  at  low  and  at  high  tide.  They  were 
also  found  in  the  sewage  emptying  into  the  river.  The 
spirilla  were  also  found  in  the  water  of  the  Delaware 
River  as  frequently  as  in  that  from  the  Schuylkill. 

One  hundred  and  ten  pure  cultures  of  spirilla  were  iso- 
lated from  the  sources  mentioned  and  subjected  to  routine 
tests.  It  was  found  that  few  or  none  of  them  were  iden- 
tical in  all  points.  There  seems,  therefore,  to  be  a  family 
of  river  spirilla  related  to  each  other  like  the  different 
colon  bacilli  are  related. 

1  Journal  of  Experimental  Medicine,  vol.  ii.,  No.  5,  p.  535. 


344  PATHOGENIC  BACTERIA. 

The  opinion  of  the  writers  is  that  "the  only  trust- 
worthy difference  between  many  of  these  varieties  and 
the  true  cholera  spirillum  is  the  specific  reaction  with 
serum  from  animals  immune  from  cholera,  or  by  Pfeiffer's 
method  of  intraperitoneal  testing  in  such  animals." 

In  discussing  these  spirilla  of  the  Philadelphia  waters 
Bergy !  says: 

"The  most  important  point  with  regard  to  the  occur- 
rence of  these  organisms  in  the  river  water  around  Phil- 
adelphia, is  the  fact  that  similar  organisms  have  been 
found  in  the  surface-waters  of  the  European  cities  in 
which  there  had  recently  been  an  epidemic  of  Asiatic 
cholera,  notably  at  Hamburg  and  Altona.  .  .  .  The  fore- 
most bacteriologists  of  Europe  have  been  inclined  to  the 
opinion  that  the  organisms  which  they  found  in  the  sur- 
face-waters of  the  European  cities  were  the  remains  of 
the  true  cholera  organism,  and  that  the  deviations  in  the 
morphologic  and  biologic  characters  from  those  of  the 
cholera  organism  were  brought  about  by  their  prolonged 
existence  in  water.  No  such  explanation  of  the  occur- 
rence of  the  organisms  in  Philadelphia  waters  can  be 
given. ' ' 

1  Jour,  of  the  Amer.  Med.  Assoc.,  Oct.  23,  1897. 


CHAPTER    V. 

PNEUMONIA. 

• 

THE  term  "pneumonia,"  while  generally  understood 
to  refer  to  the  lobar  disease  particularly  designated  as 
croupous  pneumonia,  is  a  vague  one,  really  comprehend- 
ing a  variety  of  inflammatory  conditions  of  the  lung 
quite  dissimilar  in  character.  This  being  true,  no  one 
should  be  surprised  to  find  that  a  single  organism  cannot 
be  described  as  "specific"  for  all.  Indeed,  pneumonia 
must  be  considered  as  a  group  of  diseases,  and  the  various 
microbes  found  associated  with  it  must  be  described  suc- 
cessively in  connection  with  the  peculiar  phase  of  the 
disease  in  which  they  occur. 

i.  Lobar  or  Croupous  Pneumonia. — The  bacterium, 
which  can  be  demonstrated  in  at  least  75  per  cent,  of  the 
cases  of  lobar  pneumonia,  which  is  now  almost  uni- 
versally accepted  as  the  cause  of  the  disease,  and  about 
whose  specificity  very  few  doubts  can  be  raised,  is  the 
pneumococcus  of  Frankel  and  Weichselbaum. 

Priority  of  discovery  in  the  case  of  the  pneumococcus 
seems  to  be  in  favor  of  Sternberg,  who  as  early  as  1880  de- 
scribed an  identical  organism  which  he  secured  from  his 
saliva.  Curiously  enough,  Pasteur  seems  to  have  cap- 
tured the  same  organism,  also  from  saliva,  in  the  same 
year.  The  researches  of  the  observers  whose  names  are 
attached  to  the  organism  were  not  completed  until  five 
years  later.  It  is  to  Frankel,  Telamon,  and  particularly 
to  Weichselbaum,  however,  that  we  are  indebted  for  the 
discovery  of  the  relation  which  the  organism  bears  to 
pneumonia. 

The  organism  (Fig.  98)  is  variable  in  its  morphology. 
When  grown  in  bouillon  it  is  oval,  has  a  pronounced  dis- 

345 


346  PATHOGENIC  BACTERIA. 

position  to  occur  in  pairs,  and  not  infrequently  forms 
chains  of  five  or  six  members,  so  that  some  have  been 
disposed  to  look  upon  it  as  a  streptococcus  (Gamaleia). 
In  the  fibrinous  exudate  from  croupous  pneumonia,  in 
the  rusty  sputum,  and  in  the  blood  of  rabbits  and  mice 
containing  them  the  organisms  are  arranged  in  pairs, 
exhibit  a  distinct  lanceolate  shape,  the  pointed  ends 
generally  approximated,  and  are  usually  surrounded  by 
a  distinct  halo  or  capsule  of  clear,  colorless,  homogeneous 
material,  thought  by  some  to  be  a  swollen  cell-wall,  by 


FlG.  98. — Diplococcus  pneumonise,  from  the  heart's  blood  of  a  rabbit ;    x  1000 
(Frank el  and  Pfeiffer). 

others  a  mucus-like  secretion  given  off  by  the  cells.  When 
grown  ordinarily  in  culture-media,  and  especially  upon 
solid  media,  the  capsules  are  absent. 

The  organism  is  without  motility,  has  no  spores,  and 
does  not  seem  to  be  able  to  resist  any  unfavorable  con- 
ditions when  grown  artificially.  It  stains  well  with  the 
ordinary  solutions  of  the  anilin  dyes,  and  gives  most 


PNEUMONIA.  •    347 

beautiful  pictures  in  blood  and  tissues  when  stained  by 
Gram's  method.  The  capsule  does  not  stain. 

To  demonstrate  the  capsule,  the  glacial  acetic  acid 
method  may  be  used.  The  cover-glass  is  spread  with  a 
thin  film  of  the  material  to  be  examined,  which  is  dried 
and  fixed  as  usual.  Glacial  acetic  acid  is  dropped  upon 
it  for  an  instant,  poured  (not  washed)  off,  and  at  once  fol- 
lowed by  anilin-water,  gentian-violet,  in  which  the  stain- 
ing continues  several  minutes.  Finally,  the  preparation 
is  washed  in  water,  and  may  be  examined  at  once  in  water 
or  mounted  in  balsam  after  drying.  The  capsules  are 
probably  more  distinct  when  the  examination  is  made  in 
water. 

The  pneumococcus  is  no  stranger  to  us;  it  may  some- 
times be  found  in  the  saliva  of  healthy  individuals,  and 
the  inoculation  of  human  saliva  into  rabbits  frequently 
causes  a  septicemia  in  which  the  bacillus  is  found  abun- 
dantly in  the  blood  and  tissues.  Because  of  its  frequent 
presence  in  the  saliva  it  was  described  by  Fliigge  as  the 
Bacillus  septicus  sputigenus. 

When  desired  for  purposes  of  study,  it  may  be  obtained 
by  inoculating  rabbits  with  pneumonic  sputum  and  re- 
covering the  organisms  from  their  heart's  blood,  or  it  may 
be  secured  from  the  rusty  sputum  of  pneumonia  by  the 
method  employed  by  Kitasato  for  securing  tubercle  ba- 
cilli from  sputum.  A  single  mouthful  of  fresh  sputum 
is  secured,  washed  in  several  changes  of  sterile  water  to 
free  it  from  bacteria  of  the  mouth  and  pharynx,  carefully 
separated,  and  a  central  portion  transferred  to  an  appro- 
priate culture-medium. 

The  organism  grows  upon  all  the  culture-media  except 
potato,  but  only  between  the  temperature-extremes  of 
24°  and  42°  C. ;  the  best  development  is  at  37°  C.  The 
growth  is  always  limited,  probably  because  the  formic 
acid  produced  serves  to  check  it.  The  addition  of  an 
unusual  amount  of  alkali  to  the  culture-medium  favors 
the  growth. 

The  organisms  readily  lose  their  virulence  in  culture- 


348  PA  THOGENIC  BA  CTERIA. 

media,  and  cease  to  be  pathogenic  after  a  few  days.  In  his 
experiments  with  antipneumococcic  serum  Washbourn 
found,  however,  that  a  pneumococcus  isolated  from  pneu- 
monia sputum  and  passed  through  one  mouse  and  nine 
rabbits  developed  a  permanent  virulence  when  kept  on 
agar-agar  made  carefully,  so  that  it  was  not  heated  beyond 
100°  C.,  and  alkalinized  4  c.cm.  of  normal  caustic  soda 
solution  beyond  the  neutral  point  determined  with  rosalic 
acid,  to  each  liter.  The  agar-agar  is  first  streaked  with 
sterile  rabbit's  blood,  then  inoculated.  The  cultures  are 


FIG.  99.— Diplococcus  pneumoniae :  colony  twenty-four  hours  old  upon  gelatin ; 
x  100  (Frankel  and  Pfeiffer). 

kept  at  37.5°  C.  Not  only  is  this  true,  but  ordinarily 
they  seem  to  be  unable  to  accommodate  themselves  to  a 
purely  saprophytic  life,  and  unless  continually  trans- 
planted to  new  media  die  in  a  week  or  two,  sometimes 
sooner. 

Kinyoun  recommended  to  the  writer  that  virulence 
could  be  retained  for  a  considerable  time  by  keeping 
blood  from  an  infected  rabbit,  in  a  hermetically  sealed 
glass  tube,  on  ice.  This  plan  seems  to  work  admirably 
if  the  blood  is  not  kept  too  long. 


PNEUMONIA.  349 

The  colonies  which  develop  at  24°  C.  upon  15  per 
cent,  gelatin  plates  are  described  as  small,  round,  cir- 
cumscribed, finely  granular  white  points  which  grow 
slowly,  never  attain  any  considerable  size,  and  do  not 
liquefy  the  gelatin  (Fig.  99). 

If,  instead  of  gelatin,  agar-agar  be  used  and  the  plates 
kept  at  the  temperature  of  the  body,  the  colonies  which 
develop  upon  the  plates  appear  as  transparent,  delicate, 
drop-like  accumulations,  scarcely  visible  to  the  naked 
eye,  but  under  the  microscope  distinctly  granular,  the 
central  darker  portion  being  frequently  surrounded  by  a 
paler  marginal  zone. 

In  gelatin  puncture-cultures,  made  with  15  instead  of 
the  usual  10  per  cent,  of  gelatin,  the  growth  takes  place 
along  the  entire  path  of  the  wire  in  the  form  of  little 
whitish  granules  distinctly  separated  from  each  other. 
The  growth  in  gelatin  is  always  very  limited. 

Upon  agar-agar  and  blood-serum  the  growth  consists 
of  minute,  transparent,  semi-confluent,  colorless,  dew- 
drop-like  colonies,  which  die  before  attaining  a  size 
which  permits  of  their  being  seen  without  careful  in- 
spection. 

In  bouillon  the  organisms  grow  well,  clouding  the 
medium  very  slightly. 

Milk  is  quite  well  adapted  as  a  culture-medium,  its 
casein  being  coagulated. 

No  growth  can  be  secured  upon  potato  at  any  tem- 
perature or  by  any  manipulation  yet  known.1 

When  it  is  desired  to  maintain  or  increase  the  virulence 
of  a  culture  it  must  be  very  frequently  passed  through 
the  body  of  a  rabbit.  The  degree  to  which  the  virulence 
can  be  raised  in  this  way  is  remarkable.  C.  W.  Lincoln 
has  succeeded  in  reducing  the  fatal  dose  for  rabbits  to 

TTO-wiTOTo  °f  a  c.cm. 

If  a  small  quantity  of  a  pure  culture  of  the  virulent 

1  Ortmann  asserts  that  the  pneumococcus  can  be  grown  on  potato  at  37°  C., 
hut  this  is  not  generally  confirmed.  The  usual  acid  reaction  of  the  potato 
would  indicate  that  it  was  a  very  unsuitable  culture-medium. 


350  PATHOGENIC  BACTERIA. 

organism  is  introduced  into  a  mouse,  rabbit,  or  guinea- 
pig,  the  animal  dies  in  one  or  two  days.  Exactly  the 
same  result  can  be  obtained  by  the  introduction  of  a 
piece  of  the  lung-tissue  from  croupous  pneumonia,  by 
the  introduction  of  some  of  the  rusty  sputum,  and  gener- 
ally by  the  introduction  of  saliva. 

The  post-mortem  shows  that  an  inflammatory  change 
has  taken  place  at  the  point  of  inoculation,  with  a  fibrin- 
ous  exudate  resembling  somewhat  that  in  diphtheria. 
At  times,  and  especially  in  dogs,  there  may  be  a  little 
pus  formed.  The  other  appearances  are  those  of  a 
general  disturbance.  The  spleen  is  much  enlarged,  is 
firm  and  red  brown.  The  blood  in  all  the  organs  contains 
large  numbers  of  the  bacteria,  most  of  which  exhibit  a 
distinct  lanceolate  form  and  have  their  capsules  very 
distinct.  The  disease  is  a  pure  septicemia  unassociated 
with  pronounced  tissue-changes. 

In  cases  of  the  kind  described  the  lungs  show  no  pneu- 
monic changes.  Likewise,  if  the  hypodermic  needle 
used  for  injection  be  plunged  through  the  breast-wall 
into  the  pulmonary  tissue,  no  pneumonia  results.  Mon- 
ti, however,  claims  to  have  found  that  a  true  character- 
istic pneumonia  results  from  the  injection  of  cultures 
into  the  trachea  of  susceptible  animals.  This  observa- 
tion lacks  confirmation. 

Not  all  animals  are  susceptible.  Guinea-pigs,  mice, 
and  rabbits  are  highly  sensitive  to  the  operations  of  the 
organism  ;  dogs  are  comparatively  immune. 

From  this  brief  review  of  the  peculiarities  of  the  pneu- 
mococcus  it  must  be  obvious  that  its  reputation  in  pneu- 
monia depends  more  upon  the  regularity  with  which  it  is 
found  in  that  disease  than  upon  its  capacity  to  produce  a 
similar  affection  in  the  lower  animals. 

As  in  numerous  other  diseases,  we  are  unable  to  furnish 
an  absolute  proof  of  specificity  according  to  the  postu- 
lates of  Koch. 

The  disease  is  peculiar  in  that  recovery  from  it  is  fol- 
lowed either  by  no  immunity  or  by  one  of  such  brief  dura- 


PNEUMONIA.  351 

tion  as  to  allow  of  frequent  relapses  ;  and  it  is  well  known 
that  many  cases  show  a  subsequent  predisposition  to 
fresh  attacks  of  the  disease.  This  brevity  of  immunity 
lessens  the  probability  that  in  the  future  we  shall  dis- 
cover an  antitoxin  that  shall  be  powerful  in  its  influ- 
ence upon  the  course  and  termination  of  the  disease. 

The  experiments  of  G.  and  F.  Klemperer,  a  few  years 
ago,  showed  that  the  serum  of  immunized  rabbits  pro- 
tected animals  inoculated  with  the  pneumococcus.  The 
principle  failed,  however,  when  applied  to  human  medi- 
cine. The  treatment  of  pneumonia  by  the  injection  of 
blood-serum  from  convalescents  has  also  been  abandoned 
as  useless  and  dangerous. 

Washbourn  has  recently  prepared  an  antipneumococcic 
serum  which  is  efficacious  in  protecting  rabbits  against 
ten  times  the  fatal  dose  of  live  pneumococci.  In  general, 
the  lines  upon  which  he  operated  were  those  of  Behring, 
Marmorek's  work  with  the  streptococcus  furnishing  most 
of  the  details.  A  pony  was  subjected  to  immunization 
for  a  period  of  five  months,  allowed  to  rest  three  or  four 
months  until  the  live  pneumococci  introduced  were  all 
destroyed,  and  then  bled.  Two  cases  of  human  pneu- 
monia seem  to  have  received  some  benefit  from  the  injec- 
tion of  large  doses  of  this  serum. 

The  pneumococcus  causes  other  lesions  than  croupous 
pneumonia;  thus,  Foa,  Bordoni-Uffreduzzi,  and  others 
have  found  it  in  cerebrospinal  meningitis;  Frankel,  in 
pleuritis;  Weichselbaum,  in  peritonitis;  Banti,  in  peri- 
carditis; numerous  observers  have  found  it  in  acute  ab- 
scesses; Gabbi  has  isolated  it  from  a  case  of  suppurative 
tonsillitis;  Axenfeld  has  observed  an  epidemic  of  con- 
junctivitis caused  by  it;  and  Zaufal,  Levy,  and  Schrader 
and  Netter  have  been  able  to  demonstrate  its  presence  in 
the  pus  of  otitis  media.  It  has  also  been  reported  as  oc- 
curring in  the  joints  in  arthritis  following  pneumonia. 

The  pneumococcus  is  often  present  in  the  mouths  of 
healthy  persons.  The  conditions  under  which  it  enters 
the  lung  to  produce  pneumonia  are  not  known. 


352  PATHOGENIC  BACTERIA. 

In  the  opinion  of  most  authorities,  something  more 
than  the  simple  entrance  of  the  bacterium  into  the  lung 
is  required  for  the  production  of  the  disease,  but  what 
that  something  is,  is  still  a  matter  of  doubt.  It  would 
seem  to  be  some  systemic  depravity,  and  in  support  of  this 
view  we  may  point  out  that  pneumonia  is  very  frequent, 
and  almost  universally  fatal,  among  drunkards.  Whether, 
however,  any  vital  depression  or  systemic  depravity  will 
predispose  to  the  disease,  or  whether  it  depends  for  its 
origin  upon  the  presence  of  a  certain  leucomaine,  time 
and  further  study  will  be  required  to  tell 

Bacillus  Pneumonic?  of  Friedlander  (Fig.  100). — An  un- 


FlG.   100.— Bacillus  pneumonia?  of  Friedlander,  from  the   expectoration   of  a 
pneumonia  patient;    x  1000  (Frankel  and  Pfeiffer). 

fortunate  accident  has  applied  the  name  "pneumococcus" 
to  an  organism  very  different  from  the  one  just  described. 
It  was  discovered  by  Friedlander  in  1883  in  the  exudate 
from  the  Inner  in  croupous  pneumonia,  and,  being  thought 
by  its  discoverer  to  be  the  cause  of  the  disease,  very  natu- 
rally was  called  the  pneumococcus,  or,  more  correctly,  the 
pufinnotnu-illiis.  The  grounds  upon  which  the  pathog- 
en v  of  the  organism  was  supposed  to  depend  were  very  in- 
sufficient, and  the  bacillus  of  Friedlander— or,  as  Fliigge 


PNEUMONIA.  353 

prefers  to  call  it,  the  Bacillus  pneumoniae — has  ceased  to 
be  regarded  as  specific,  and  is  now  looked  upon  as  an 
accidental  organism  whose  presence  in  the  lung  is,  in 
most  cases,  unimportant. 

As  the  two  organisms  are  similar  in  more  respects  than 
their  names,  Friedlander's  bacillus  requires  at  least  a 
brief  description. 

It  is  distinctly  a  bacillus,  but  sometimes,  when  occur- 
ring in  pairs,  has  a  close  resemblance  to  the  pneumo- 
coccus  of  Frankel  and  Weichselbaum.  Very  frequently 
it  forms  chains  of  four  or  more  elements.  It  is  also  com- 
monly surrounded  by  a  transparent  capsule.  It  is  non- 
motile,  has  no  spores  and  no  flagella.  It  stains  well 
with  the  ordinary  anilin  dyes,  but  does  not  retain  the 
color  when  stained  by  Gram's  method. 

Frankel  points  out  that  Friedlander's  error  in  suppos- 
ing this  bacillus  to  be  the  chief  parasite  in  pneumonia 
depended  upon  the  fact  that  his  studies  were  made  by 
the  plate  method.  If  some  of  the  pneumonic  exudate  be 
mixed  with  gelatin  and  poured  upon  plates,  the  bacilli 
grow  into  colonies  at  the  end  of  twenty-four  hours,  and 
appear  as  small  white  spheres  which  spread  upon  the 
gelatin  to  form  white  masses  of  a  considerable  size. 
Under  the  microscope  these  colonies  are  rather  irregular 
in  outline  and  somewhat  granular. 

The  bacillus  grows  at  as  low  a  temperature  as  16°  C., 
and,  according  to  Sternberg,  has  a  thermal  death-point 
of  56°  C. 

When  a  colony  is  transferred  to  a  gelatin  puncture-cul- 
ture, quite  a  massive  growth  occurs.  Upon  the  surface  a 
somewhat  elevated,  rounded  white  mass  is  formed,  and 
in  the  track  of  the  wire  innumerable  little  colonies 
spring  up  and  become  confluent,  so  that  a  "  nail-growth  " 
results.  No  liquefaction  occurs.  When  old  the  cultures 
sometimes  become  brown  in  color. 

Upon  the  surface  of  agar-agar  at  ordinary  temperatures 
quite  a  luxuriant  white  or  brownish-yellow,  smeary,  cir- 

23 


354  PATHOGENIC  BACTERIA. 

cumscribed  growth  occurs.  The  growth  upon  blood- 
serum  is  the  same. 

Upon  potato  the  growth  is  abundant,  quickly  covering 
the  entire  surface  with  a  thick  yellowish-white  layer, 
which  sometimes  contains  bubbles  of  gas.  Gas  is  also 
sometimes  developed  in  gelatin  cultures. 

A  most  superficial  comparison  will  suffice  to  show  the 
great  difference  in  vegetation  between  these  two  so-called 
pneumococci. 

Friedlander  had  considerable  difficulty  in  causing  any 
pathogenic  changes  by  the  injection  of  his  bacillus  into 
animals.  Rabbits  and  guinea-pigs  were  immune,  and 
the  only  actual  pathogenic  results  which  Friedlander  ob- 
tained were  in  mice,  into  whose  lungs  and  pleura  he 
injected  the  cultures.  The  remarks  of  Frankel  upon 
such  mouse-operations,  which  do  not  add  much  weight 
to  experiments,  have  already  been  quoted. 

In  the  status  prcesens  of  bacteriologic  knowledge  the 
bacillus  of  Friedlander  is  regarded  as  an  organism  of  very 
feeble  pathogenic  powers,  generally  a  harmless  sapro- 
phyte, but  which  may  at  times  aid  in  producing  inflam- 
matory changes  when  in  the  tissues  of  the  human  body. 

2.  Catarrhal   Pneumonia. — This  form  of  pulmonary 
inflammation   occurs   in   local   areas,   generally  situated 
about   the   distribution  of  a  bronchiole.     It   cannot   be 
said  to  have  a  specific   micro-organism,  as   almost  any 
irritant  foreign  materials  accidentally  inhaled  can  cause 
it.     The  majority  of  the  cases,  however — and  especially 
those  which  are  distinctly  peribronchial — are  caused  by 
the  presence  of  the  staphylococcus  and  streptococcus  of 
suppuration.     Friedlander' s  bacillus  may  also  aid  in  pro- 
ducing local  inflammations. 

3.  Tubercular  Pneumonia. — At  times  the  process  of 
pulmonary  tuberculosis  is  so  rapid,  and  associated  with 
the   production  of    so   much   semi-liquid,   semi-necrotic 
material,  that  the  auto-infection  of  the  lung  is  greatly 
favored ;  the  tubercle  bacilli  are  distributed  to  the  entire 
lung  or  to  large  parts  of  it,  and  a  distinct  inflammation 


PNEUMONIA.  355 

occurs.  Such  a  pneumonia  may  be  caused  by  the  tubercle 
bacillus  alone,  but  more  often  it  is  aided  by  accompany- 
ing staphylococci,  streptococci,  tetragenococci,  pneumo- 
cocci,  pneurnobacilli,  and  other  organisms  apt  to  be  pres- 
ent in  a  lung  in  which  tuberculosis  is  in  progress  and 
ulceration  and  cavity-formation  are  advanced. 

4.  Mixed  Pneumonias. — It  frequently  happens  that 
pneumonia  occurs  in  the  course  of,  or  shortly  after  the 
convalescence  from,  influenza.  In  these  cases  a  mixed 
infection  is  present,  and  there  is  no  difficulty  in  deter- 
mining that  both  the  influenza  bacillus  and  the  pneumo- 
coccus  are  present.  Again,  sometimes  the  pneumococci 
and  staphylococci  operate  simultaneously,  and  produce 
a  purulent  pneumonia  with  abscesses  as  the  conspicuous 
feature.  As  almost  any  combination  of  the  described 
bacteria  is  possible  in  the  lungs,  and  as  these  combi- 
nations will  all  produce  varying  inflammatory  conditions, 
it  must  be  left  for  the  student  to  imagine  what  the  par- 
ticular characters  of  each  may  be. 

Among  these  mixed  pneumonias  may  be  mentioned 
those  called  by  Klemperer  and  Levy  "complicating 
pneumonias,"  occurring  in  the  course  of  typhoid,  etc. 


C.     THE   SEPTIC   DISEASES. 


CHAPTER    I. 
ANTHRAX. 

THE  disease  of  cattle  known  as  anthrax  or  "splenic 
fever  "  is  of  infrequent  occurrence  in  this  country  and  in 
England.  In  France,  Germany,  Hungary,  Russia,  Persia, 
and  the  East  Indian  countries  it  is  a  dreaded  and  common 
malady  which  robs  herdsmen  of  many  of  their  valuable 
stock.  Siberia  perhaps  suffers  most,  the  disease  being  so 
exceedingly  common  and  malignant  as  to  deserve  the 
name  "  Siberian  pest."  Certain  local  areas,  such  as  the 
Tyrol  and  Auvergne,  in  which  it  seems  to  be  constantly 
present,  serve  as  distributing  foci  from  which  the  disease 
spreads  rapidly  in  summer,  afflicting  many  animals,  and 
ceasing  its  depredations  only  with  the  advent  of  winter. 
It  seems  to  be  distinctly  a  disease  of  the  summer  season. 

The  animals  most  frequently  affected  are  cows  and 
sheep.  Among  our  laboratory  animals  white  mice, 
guinea-pigs,  and  rabbits  are  highly  susceptible ;  dogs, 
cats,  most  birds,  and  amphibians  are  almost  perfectly 
immune.  White  rats  are  infected  with  difficulty.  Man 
is  only  slightly  susceptible,  the  manifestation  of  the  dis- 
ease as  seen  in  the  human  species  being  different  from 
the  same  disease  in  the  lower  animals  in  that  it  is  usually 
a  local  affection — malignant  carbuncle — and  only  at  times 
gives  rise  to  a  general  infection. 

Anthrax  was  one  of  the  first  of  the  specific  diseases 
proven  to  be  caused  by  a  definite  micro-organism.  As 
early  as  1849,  Pollender  discovered  small  rod-shaped 
bodies  in  the  blood  of  animals  suffering  from  anthrax, 
but  the  exact  relation  which  they  bore  to  the  disease  was 
not  pointed  out  until  1863,  when  Davaine,  by  a  series  of 
interesting  experiments,  proved  to  most  unbiased  minds 
their  etiological  significance.  The  further  confirmation 

356 


ANTHRAX.  357 

of  Davaine's  conclusions  and  actual  proof  of  the  matter 
rested  with  Pasteur  and  Koch,  who,  observing  that  the 
bacilli  bore  spores,  cultivated  them  successfully  outside 
the  body,  and  then  produced  the  disease  by  the  inocula- 
tion of  pure  cultures. 

The  anthrax  bacilli  (Fig.  101)  are  large  rods  with  a 


FIG.  101. — Bacillus  anthracis  :  colony  three  days  old  upon  a  gelatin  plate;  ad- 
hesive preparation ;  x  looo  (Frankel  and  Pfeiffer). 

rectangular  form,  caused  by  the  very  slight  rounding  of 
the  corners.  They  measure  5-20  //  in  length  and  are 
from  i  fJL  to  1.25  //  in  breadth.  The  pronounced  tendency 
is  toward  the  formation  of  long  threads,  in  which,  how- 
ever, the  individuals  can  generally  be  made  out ;  at  times 
isolated  rods  occur.  In  the  threads  the  bacilli  seem  en- 
larged a  little  at  the  ends,  and  give  somewhat  the  appear- 
ance of  a  bamboo  cane.  The  formation  of  spores  is  pro- 
lific :  each  spore  has  a  distinct  oval  shape,  is  transparent, 
and  does  not  alter  the  contour  of  the  bacillus  in  which  it 
occurs.  Spores  are  generally  formed  in  the  presence  of 
oxygen  upon  the  surfaces  of  the  culture-media.  When  a 
spore  is  placed  under  favorable  conditions  for  its  devel- 
opment and  is  carefully  watched,  it  may  be  observed  to 
increase  in  length  a  trifle,  then  to  undergo  a  rupture  at 


358  PATHOGENIC  BACTERIA. 

one  end,  from  which   the  new  bacillus  projects.      The 
spores  of  anthrax  (Fig.  102),  being  large  and  easily  ob- 


, 

•  % 


*'***-'  5 . 

M'' 


FIG.  102. — Bacillus   anthracis,  stained  to  show  the  spores;  x  icoo  (Frankel 

and  Pfeiffer). 

tainable,  are  excellent  subjects  for  the  study  of  sporula- 
tion,  for  the  action  of  germicides  and  antiseptics,  and  for 
demonstration  by  stains.  When  dried  upon  threads  of 
silk  they  will  retain  their  vitality  for  several  years,  and 
are  highly  resistant  to  heat  and  disinfectants. 

Spores  of  anthrax  are  killed  by  five  minutes'  exposure 
to  a  temperature  of  100°  C.,  and  are  killed  in  five  minutes 
in  a  5  per  cent,  solution  of  carbolic  acid,  or,  at  least,  are 
deprived  of  their  vegetative  property  in  relation  to  cul- 
ture-media. It  is  said  by  some  that  spores  subjected  to 
5  per  cent,  carbolic  acid  can  germinate  when  introduced 
into  susceptible  animals.  Spores  are  also  killed  by  simple 
wetting  with  i  :  100,000  bichlorid-of-mercury  solution. 

The  bacilli  are  not  motile  and  are  not  provided  with 
flagella.  They  stain  well  with  ordinary  solutions  of  the 
anilin  dyes,  and  can  be  beautifully  demonstrated  in  the 
tissues  by  Gram's  method  and  by  Weigert's  fibrin  method. 
Picro-carmin,  followed  by  Gram's  method,  gives  a  beauti- 
ful, clear  picture.  The  spores  can  be  stained  with  carbol- 


ANTHRAX.  359 

fuchsin,  the  bacilli  decolorized  with  a  very  weak  acid  and 
then  counter-stained  with  a  watery  solution  of  methyl  blue. 
Upon  the  surface  of  gelatin  plate-cultures  the  bacillus 
forms  beautiful  and  highly  characteristic  colonies  (Fig. 
103).  To  the  naked  eye  they  appear  first  as  minute 


FIG.  103. — Bacillus  anthracis:  colony  upon  a  gelatin  plate  ;  x  100  (Frankel  and 

Pfeiffer). 

round  whitish  dots  occurring  upon  the  surface,  and  caus- 
ing liquefaction  of  the  gelatin  as  they  increase  in  size. 
Under  the  microscope  they  can  be  seen  in  the  gelatin  as 
egg-shaped,  slightly  brownish  granular  bodies,  not  attain- 
ing their  full  development  except  upon  the  surface,  where 
they  spread  out  into  flat,  irregular,  transparent  growths 
bearing  a  partial  resemblance  to  tufts  of  curled  wool. 
From  a  tangled  centre  large  numbers  of  curls  extend, 
each  made  up  of  parallel  threads  of  bacilli.  As  soon  as 
the  colony  attains  any  considerable  size  liquefaction  be- 
gins. These  colonies  make  beautiful  adhesive  prepara- 
tions. If  a  perfectly  clean  cover-glass  be  passed  once 
through  a  flame  and  laid  carefully  upon  the  gelatin,  the 
colonies  can  generally  be  picked  up  entire  when  the  glass  is 
removed.  Such  a  specimen  can  be  dried,  fixed,  and  stained 
in  the  same  manner  as  an  ordinary  cover-glass  preparation. 


360  PATHOGENIC  BACTERIA. 

In  gelatin  puncture-cultures  the  growth  is  even  more 
characteristic  than  are  the  colonies.  The  bacilli  begin 
to  grow  along  the  entire  track  of  the  wire,  most  luxuri- 
antly at  the  surface,  where  oxygen  is  plentiful.  As  the 
growth  progresses  fine  filaments  like  bristles,  extend 
from  the  puncture  into  the  neighboring  gelatin  giving 
the  growth  somewhat  the  appearance  of  an  evergreen 
tree  inverted  (Fig.  104). 


FIG.  104. — Bacillus  anthracis  :  gelatin  puncture-culture  seven  days  old 
(Gunther). 

The  more  superficial  of  these  threads  reach  about  half- 
way to  the  sides  of  the  tube,  while  the  deeper  ones  are 
shorter  and  shorter,  until  near  the  apex  branches  cease. 
When  the  projections  are  pretty  well  developed  a  distinct 
surface-growth  will  be  discerned,  and  if  the  tube  be  tilted, 
one  can  observe  that  the  gelatin  beneath  it  has  liquefied. 
As  the  growth  becomes  older  the  liquefaction  increases, 
until  ultimately  the  entire  gelatin  is  fluid  and  the  growth 
is  precipitated. 

Upon  agar-agar  the  characteristics  are  few.  The 
growth  takes  place  all  along  the  line  of  inoculation  as 
a  slightly  translucent,  slightly  wrinkled  layer  with  irreg- 
ular edges,  from  which  sufficient  bacillary  threads  pro- 
ject to  give  it  a  ciliated  appearance  to  the  naked  eye. 
When  the  culture  is  old  the  agar-agar  turns  a  distinct 
brown.  Spore-formation  is  luxuriant  upon  agar-agar. 


ANTHRAX.  361 

On  potato  the  growth  is  white,  creamy,  sometimes 
rather  dry  in  appearance.  Sporulation  is  marked. 

Blood-serum  cultures  lack  peculiarities ;  the  culture- 
medium  is  slowly  liquefied. 

The  bacillus  only  grows  between  the  extremes  of  20° 
and  45°  C.,  best  at  37°  C.  The  exposure  of  the  organ- 
ism to  the  temperature  of  42-43°  C.  for  twenty-four  hours 
is  sufficient  to  destroy  its  virulence. 

The  culture-media  should  always  be  faintly  alkaline,  as 
anthrax  bacilli  will  not  grow  in  the  presence  of  free  acid. 

The  micro-organism  under  consideration  is  a  parasitic 
microbe,  yet  is  one  which,  because  of  its  spores,  can,  in 
a  latent  form,  exist  without  the  animal  organism  until 
appropriate  conditions  for  its  natural  development  are 
presented. 

Ordinarily,  the  infection  takes  place  either  through  the 
respiratory  tract  or  through  the  alimentary  canal. 

Buchner  has  shown  that  when  animals  are  allowed 
to  inhale  anthrax  spores  they  die  of  typical  anthrax. 
The  spores  establish  themselves  in  the  alveoli  of  the 
lung,  penetrate  the  epithelium,  enter  the  vascular  sys- 
tem, and  soon  give  rise  to  typical  lesions.  Strange  to 
say,  the  appearance  caused  by  the  inhalation  of  the 
bacilli  in  their  perfect  form  is  entirely  different,  for  a 
rapid  multiplication  occurs  without  Sporulation,  and 
causes  a  violent  irritative  pneumonia  with  serous  or  sero- 
fibrinous  exudate  in  which  large  numbers  of  the  bacilli 
occur.  In  these  cases  there  may  be  no  general  infection. 

When  the  bacilli  are  taken  into  the  stomach  in  food 
they  meet  with  a  rapid  death  because  of  the  acidity  of 
the  gastric  juice.  Should  spores,  however,  be  ingested, 
they  are  able  to  endure  the  gastric  juice,  to  pass  into  the 
intestine,  and,  as  soon  as  proper  conditions  of  alkalinity 
are  encountered,  to  develop  into  bacilli.  They  develop 
rather  rapidly,  surround  the  villi  with  thick  networks 
of  bacillary  threads,  separate  the  epithelial  cells,  enter 
the  lymphatics,  and  thus  find  the  appropriate  environ- 
ment for  the  production  of  a  general  infection. 


362  PA  THOGENIC  BACTERIA . 

Sometimes  the  bacillus  enters  the  body  through  a 
wound,  cut,  scratch,  or  fly-bite.  This  is  especially  the 
case  with  men  who  come  in  contact  with  diseased  cattle. 
As  has  already  been  pointed  out,  a  malignant  pustule 
is  apt  to  follow,  and  may  cause  death.  Men  whose 
occupations  bring  them  in  contact  with  skins  and  hair 
from  animals  dead  of  anthrax  are  not  only  liable  to 
wound-infection,  but  are  sometimes  the  subjects  of  a  pul- 
monary form  of  the  disease — " wool-sorter's  disease" 
caused  by  inspiration  of  the  spores  attached  to  the  wool. 

The  disease  as  we  see  it  in  the  laboratory  is  accom- 
panied by  few  but  marked  lesions.  The  ordinary  method 
of  inoculation  is  to  cut  away  a  little  of  the  hair  from 
the  abdomen  of  a  guinea-pig  or  rabbit  or  the  root  of 
a  mouse's  tail,  make  a  little  subcutaneous  pocket  with 
a  snip  of  a  pair  of  sterile  scissors,  and  introduce  the 
spores  or  bacilli  from  a  pure  culture  upon  a  rather  heavy 
platinum  wire,  the  end  of  which  is  flattened,  pointed, 
and  perforated.  An  animal  inoculated  in  this  way  gen- 
erally dies,  according  to  the  species,  in  from  twenty-four 
hours  to  three  days.  The  symptoms  are  weakness,  fever, 
loss  of  appetite,  and  sometimes  a  bloody  discharge  from 
nose  and  bowels.  There  is  much  subcutaneous  edema. 
At  the  autopsy  very  little  change  is  observed  at  the  seat 
of  inoculation.  The  subcutaneous  tissue  beneath  it  for 
a  considerable  distance  around  is  occupied  by  a  peculiar 
colorless  gelatinous  edema  which  contains  the  bacilli. 
The  abdominal  cavity  shows  injection  and  congestion 
of  its  viscera.  The  spleen  is  considerably  enlarged,  is 
dark  in  color,  and  of  mushy  consistence.  The  liver  is 
somewhat  enlarged.  When  the  thorax  is  opened,  the 
lunjrs  may  be  slightly  congested,  but  otherwise  no 
changes  are  to  be  found. 

When  the  various  organs,  which  present  no  appreciable 
changes  to  the  naked  eye,  are  subjected  to  a  microscopic 
examination,  the  appropriate  staining  methods  bring  out 
a  most  remarkable  and  beautiful  change.  The  capil- 
lary system  is  almost  universally  occupied  by  bacilli, 


ANTHRAX.  363 

which  extend  throughout  its  meshworks  in  long  threads. 
Most  beautiful  bundles  of  these  bacillary  threads  can,  at 
times,  be  found  in  the  glomeruli  of  the  kidney  and  in 
the  minute  capillaries  of  the  intestinal  villi.  In  the 
larger  vessels,  where  the  blood-stream  is  rapid,  the  bac- 
teria are  relatively  few,  so  that  the  burden  of  bacillary 
obstruction  is  borne  by  the  minute  vessels.  The  con- 
dition is  thus  one  of  pure  septicemia,  and  bacilli  can  be 
secured  in  pure  cultures  from  the  blood  and  tissues. 

The  susceptibility  of  the  anthrax  bacillus  to  the  influ- 
ence of  heat,  cold,  antiseptics,  etc.  not  only  permitted 
Buchner,  Behring,  and  others  to  produce  biological  curi- 
osities in  the  form  of  bacilli  unable  to  bear  spores  and 
robbed  of  their  pathogenic  powers,  but  also  suggested 
to  Pasteur  the  important  practical  measure  of  protective 
vaccination.  Pasteur  found  that  the  inoculation  of  non- 
virulent  bacilli  into  cows  and  sheep,  and  their  reinocula- 
tion  with  slightly  virulent  bacilli,  gave  them  the  ability 
to  withstand  the  action  of  highly  virulent  organisms. 
Loffler,  Kochy  and  Gaffky,  however,  found  that  these 
immunized  animals  were  not  absolutely  protected  from 
intestinal  anthrax. 

The  methods  of  diminishing  the  virulence  of  the 
anthrax  bacilli  are  numerous.  Toussaint,  who  was  cer- 
tainly the  first  to  produce  immunity  in  animals  by  inject- 
ing them  with  sterile  cultures  of  the  bacillus,  found  that 
the  addition  of  i  per  cent,  of  carbolic  acid  to  blood  of 
animals  dead  of  anthrax  destroyed  the  virulence  of  the 
bacilli ;  Chamberland  and  Roux  found  it  removed  when 
o.  1-0.2  per  cent,  of  bichromate  of  potassium  was  added  to 
the  culture-medium  ;  Chauveau  used  atmospheric  pressure 
to  the  extent  of  six  to  eight  atmospheres  and  found  the 
virulence  diminished  ;  Arloing  found  that  direct  sunlight 
operated  similarly ;  Lubarsch  found  that  the  inoculation 
of  the  bacilli  into  immune  animals,  such  as  the  frog,  and 
their  subsequent  recovery  from  its  blood,  diminishes  the 
virulence  markedly. 

Protection  can  be  afforded  in  still  other  ways.     The 


364  PATHOGENIC  BACTERIA. 

simultaneous  inoculation  of  bacteria  not  at  all  related  to 
anthrax  will  sometimes  recover  the  animal,  as  Htippe 
found.  Hankin  found  in  the  cultures  chemical  sub- 
stances, especially  an  albuminose,  which  exerted  a  pro- 
tective influence.  Chamberland  has  shown  that  pro- 
tective inoculation  by  Pasteur's  method  has  diminished 
the  death-rate  from  10  per  cent,  for  sheep  and  5  per 
cent,  for  cattle  to  about  0.94  per  cent,  for  sheep  and  0.34 
per  cent,  for  cattle,  so  that  the  utility  of  the  method  is 
scarcely  questionable.  In  1890,  Ogata  and  Jasuhara 
showed  that  in  the  convalescents  from  anthrax  among 
their  experimental  animals  an  antitoxic  substance  was 
present  in  the  blood  in  such  quantities  that  i  :  800  parts 
per  body- weight  of  dog's  serum  containing  the  antitoxin 
would  protect  a  mouse.  Similar  results  have  been  at- 
tained by  Marchoux. 

Experiments  of  interest  have  been  performed  to  show 
that  the  natural  immunity  enjoyed  by  many  animals  can 
be  destroyed.  Behring  found  that  if  the  alkalinity  of  the 
blood  of  rats  was  diminished,  they  could  become  affected 
with  anthrax,  and  numerous  observers  have  shown  that 
when  anthrax  bacilli  and  unrelated  organisms,  such  as 
the  erysipelas  cocci,  Bacillus  prodigiosus,  and  Bacillus 
pyocyaneus,  are  simultaneously  introduced  into  immune 
animals,  the  immunity  is  destroyed  and  the  animals 
succumb  to  the  disease.  Frogs  have  been  made  to  suc- 
cumb to  the  disease  by  exposure  to  a  temperature  of  37° 
C.  after  inoculation.  Pasteur  destroyed  the  immunity  of 
fowls  by  a  cold  bath  after  inoculation. 

In  the  natural  order  of  events  anthrax  in  cattle  is 
probably  the  result  of  the  inhalation  or  ingestion  of  the 
spores  of  the  bacilli  from  the  pasture.  At  one  time 
much  discussion  a.rose  concerning  the  infection  of  the 
pasture.  It  was  argued  that,  the  bacilli  being  enclosed 
in  the  tissues  of  the  diseased  animals,  the  infection  of 
the  pasture  must  be  due  to  the  distribution  of  the  germs 
from  the  buried  cadaver  to  all  parts  of  the  field,  either 
through  the  activity  of  earth-worms,  which  ate  of  the 


ANTHRAX.  365 

earth  surrounding  the  corpse  and  then  deposited  the 
spores  in  their  excrement  at  remote  areas  (Pasteur),  or  to 
currents  of  moisture  in  the  soil.  Koch  seems,  however, 
to  have  demonstrated  the  fallacy  of  the  theories  by  show- 
ing that  the  conditions  under  which  the  bacilli  find  them- 
selves in  buried  cadavers  are  exactly  opposed  to  those 
favorable  to  fructification  or  sporulation,  and  that  in  all 
probability  the  majority  of  bacteria  suffer  the  same  fate 
as  the  animal  cells,  and  disintegrate,  especially  if  the  ani- 
mal be  buried  at  a  depth  of  two  or  three  meters. 

Frankel  points  out  particularly  that  no  infection  of  the 
soil  by  the  dead  animal  could  be  worse  than  the  pollution 
of  its  surface  by  the  bloody  stools  and  urine,  rich  in 
bacilli,  discharged  upon  it  by  the  animal  before  death, 
and  that  in  all  probability  it  is  the  live,  and  not  the  dead, 
animals  that  are  to  be  blamed  as  sources  of  infection. 

As  every  animal  affected  with  anthrax  is  a  source  of 
danger  to  the  community  in  which  it  lives,  to  the  men 
who  handle  it  as  well  as  the  animals  who  browse  beside 
it,  such  animals,  as  soon  as  the  diagnosis  is  made,  should 
be  killed,  and,  together  with  the  hair  and  skin,  be  burned. 
When  this  is  impracticable,  Frankel  recommends  that 
they  be  buried  to  a  depth  of  at  least  iJ^-2  meters,  so 
that  the  sporulation  of  the  bacilli  is  impossible.  The 
dejecta  should  also  be  carefully  disinfected  with  5  per 
cent,  carbolic-acid  solution. 

Of  course,  animals  can  be  infected  through  wounds. 
This  mode  of  infection  is,  however,  more  common 
among  men,  who  suffer  from  the  local  disease  mani- 
fested as  the  malignant  carbuncle,  than  among  animals. 

Occasionally  bacilli  are  encountered  presenting  all  the 
morphological  and  cultural  characteristics  of  the  anthrax 
bacillus,  but  devoid  of  any  disease-producing  power — 
Bacillus  anthracoides,  etc.  Exactly  what  relation  they 
may  bear  to  the  anthrax  bacillus  is  uncertain.  They 
may  be  entirely  different  organisms,  or  they  may  be  in- 
dividuals whose  pathogeny  has  been  lost  through  unfa- 
vorable environment. 


CHAPTER  II. 
TYPHOID   FEVER. 

THE  bacillus  of  typhoid  fever  (Fig.  105)  was  discovered 
by  Eberth  and  Koch  in  1880,  and  was  first  secured  in 


^***^SMB^^^^ 

FIG.    105. — Bacillus   typhi,   from   a   twenty-four-hours-old   agar-agar   culture; 

x  650  (Heim). 

pure   culture   from   the   spleen   and   affected    lymphatic 
glands  by  Gaffky  four  years  later. 

The  organism  is  a  small,  short  bacillus  about  1-3  /* 
(2-4  /*  Chantemesse,  Widal)  in  length  and  0.5-0.87*  broad 
(Sternberg).  The  ends  are  rounded,  and  it  is  rather  ex- 
ceptional for  the  bacilli  to  be  united  in  chains,  though 
this  arrangement  is  common  in  potato  cultures.  The 
size  and  morphology  vary  distinctly  with  the  nature  of 
the  culture-medium  and  the  age  of  the  culture.  Thoinot 
and  Masselin  in  describing  these  morphological  peculi- 
arities mention  that  when  grown  in  bouillon  it  is  a  very 
slender  bacillus ;  in  milk  it  is  a  large  bacillus ;  upon 
a^ar-a<;ar  and  potato  it  is  very  thick  and  short ;  and  in 

old  gelatin  cultures  it  forms  very  long  filaments. 
Ml 


TYPHOID  FEVER. 


367 


The  organisms  are  actively  motile,  the  motility  prob- 
ably being  caused  by  the  numerous  flagella  with  which 
the  bacilli  are  provided.  The  flagella  stain  well  by 
Loffler's  method,  and,  as  they  are  numerous  (ten  to 
twenty)  and  readily  demonstrable,  the  typhoid  bacillus  is 
the  favorite  subject  for  their  study.  The  movements  of 


FIG.  106. — Bacillus  typhi,  from  an  agar-agar  culture  six  hours  old,  showing  the 
flagella  stained  by  Loffler's  method;    x  1000  (Frankel  and  Pfeiffer). 

the  short  bacilli  are  oscillating,  those  of  the  longer  indi- 
viduals serpentine. 

The  organism  stains  quite  well  by  the  ordinary  meth- 
ods, but  loses  the  color  entirely  when  stained  by  Gram's 
method.  Its  peculiarity  of  staining  is  the  readiness  with 
which  the  bacillus  gives  up  its  color  in  the  presence  of 
solvents,  so  that  it  is  particularly  difficult  to  stain  it  in 
tissue. 

When  sections  are  to  be  stained  the  best  method  is  to 
allow  the  tissue  to  remain  in  Loffler's  alkaline  methylene 
blue  for  from  fifteen  minutes  to  twenty-four  hours,  then 
wash  in  water,  dehydrate  rapidly  in  alcohol,  clear  up  in 
xylol,  and  mount  in  Canada  balsam.  Ziehl's  method 
also  gives  good  results.  The  sections  are  stained  for  fif- 
teen minutes  in  a  solution  of  distilled  water  100,  fuch- 


368  PATHOGENIC  BACTERIA. 

sin  i,  and  phenol  5.  After  staining  they  are  washed  in 
distilled  water  containing  i  per  cent,  of  acetic  acid, 
dehydrated  in  alcohol,  cleared,  and  mounted.  In  such 
preparations  the  bacilli  may  be  found  in  little  groups, 
which  are  easily  discovered,  under  a  low  power  of 
the  microscope,  as  reddish  specks,  and  readily  resolved 
into  bacilli  with  the  high  power  of  the  oil-immersion 
lens. 

In  bacilli  stained  by  this  alkaline  methylene-blue  solu- 
tion dark-colored  dots  may  sometimes  be  observed  near 
the  ends  of  the  rods.  These  dots  were  at  first  regarded 
as  spores,  but  are  now  denominated  polar  granules,  and 
are  thought  to  be  of  no  importance. 

The  typhoid  bacillus  is  both  saprophytic  and  parasitic. 
It  finds  abundant  conditions  in  nature  for  its  growth  and 
development,  and,  enjoying  strong  resisting  powers,  can 
accommodate  itself  to  environment  much  better  than  the 
majority  of  pathogenic  bacteria,  and  can  be  found  in 
water,  air,  soiled  clothing,  dust,  sewage,  milk,  etc.  con- 
taminated directly  or  indirectly  by  the  intestinal  dis- 
charges of  diseased  persons. 

The  bacillus  is  also  occasionally  present  upon  green 
vegetables  sprinkled  with  water  containing  it,  and  epi- 
demics are  reported  in  which  the  infection  was  traced  to 
oysters  from  a  certain  place  where  the  water  was  infected 
through  sewage.  Newsholme1  found  that  in  56  cases 
of  typhoid  fever  about  one-third  was  attributable  to  the 
eating  of  raw  shell-fish.  In  such  cases  the  evidence 
accumulated  serves  to  show  that  the  shell-fish  were  from 
sewage-polluted  beds.  The  bacillus  probably  enters  milk 
occasionally  in  water  used  to  dilute  it. 

The  resistant  powers  of  the  organisms  have  already 
been  described  as  great.  They  can  grow  well  at  the 
room-temperature.  The  thermal  death-point  is  given  by 
Sternberor  as  60°  C.  The  bacilli  can,  according  to  Klem- 
perer  and  Levy,  remain  vital  for  three  months  in  distilled 
water,  though  in  ordinary  water  the  commoner  and  more 

1  Brit.  Med.  Jour.,  Jan.,  1895. 


TYPHOID  FEVER.  369 

vigorous  saprophytes  outgrow  them  and  cause  their  dis- 
appearance in  a  few  days.  When  buried  in  the  upper 
layers  of  the  soil  the  bacilli  retain  their  vitality  for  nearly 
six  months.  Robertson l  found  that  when  planted  in 
soil  and  occasionally  fed  by  pouring  bouillon  upon  the 
surface,  the  typhoid  bacillus  maintained  its  vitality  for 
twelve  months.  He  suggests  that  it  may  do  the  same 
in  connection  with  leaky  drains. 

Cold  has  no  effect  upon  typhoid  bacilli,  for  freezing 
and  thawing  several  times  are  without  injury  to  them. 
They  have  been  found  to  remain  alive  upon  linen  for 
from  sixty  to  seventy-two  days,  and  upon  buckskin  for 
from  eighty  to  eighty-five  days.  Sternberg  has  succeeded 
in  keeping  hermetically  sealed  bouillon  cultures  alive  for 
more  than  a  year.  In  the  experience  of  the  author,  un- 
less transplanted  rather  frequently,  cultures  upon  agar- 
agar  are  apt  to  die  out.  In  the  presence  of  chemical 
agents  the  bacillus  is  also  able  to  retain  its  vitality,  o.  i 
to  o.  2  per  cent,  of  carbolic  acid  added  to  the  culture- 
media  being  without  effect  upon  its  growth.  At  one 
time  the  tolerance  to  carbolic  acid  was  thought  to  be 
characteristic,  but  it  is  now  known  to  be  shared  by  other 
bacteria.  The  bacilli  seem  to  be  killed  in  a  short  time 
by  thorough  drying. 

The  bacillus  is  best  secured  in  pure  culture,  either 
from  an  enlarged  lymphatic  gland  or  from  the  splenic 
pulp  of  a  case  of  typhoid.  To  secure  the  bacillus  in  this 
way  the  autopsy  should  be  made  as  soon  after  death  as 
possible,  lest  the  Bacillus  coli  invade  the  tissue. 

Cultures  of  the  typhoid  bacillus  may  be  obtained,  but 
with  difficulty,  from  the  alvine  discharges  of  typhoid 
patients.  In  examining  this  material,  however,  it  must 
be  remembered  that  the  bacilli  are  certain  to  be  present 
only  in  the  second  and  third  weeks. 

As  numerous  saprophytic  bacteria  are  present  in  the 
feces,  the  resistance  which  the  typhoid  bacillus  exhibits 
to  carbolic  acid  can  be  made  use  of  in  obtaining  the  pure 

1  Brit.  Med.  Jour.,  Jan.  8,  1898. 
24 


370  PATHOGENIC  BACTERIA. 

culture.  To  each  of  several  tubes  of  melted  gelatin  0.05 
per  cent,  of  carbolic  acid  is  added.  This  addition  is  most 
easily  calculated  by  supposing  the  average  amount  of 
gelatin  contained  in  a  tube  to  be  10  c.cm.  To  the  aver- 
age tube  TV  c.cm.  of  a  5  per  cent,  solution  of  carbolic  acid 
is  added,  and  gives  very  nearly  the  desired  quantity.  A 
minute  portion  of  the  feces  is  broken  up  with  a  platinum 
loop  and  stirred  in  the  tube  of  melted  gelatin  ;  a  drop 
from  this  dilution  is  transferred  to  the  second  tube,  a 
drop  from  it  to  a  third,  and  then  the  contents  of  each 
tube  are  poured  upon  a  sterile  plate,  or  into  a  Petri  dish, 


Fir,.   107. — Bacillus  typhi   abdominalis:    superficial  colony  two  days  old,  as 
seen  upon  the  surface  of  a  gelatin  plate;    x  20  (Heim). 

or  rolled,  according  to  Esmarch's  plan,  in  the  manner 
already  described.  The  carbolic  acid  present  in  these 
cases  prevents  the  great  mass  of  saprophytes  from  de- 
veloping, but  allows  the  perfect  development  of  the 
typhoid  bacillus  (Fig.  107)  and  its  near  congener,  the 
Bacillus  coli  communis  (Fig.  no). 

The  colonies  that  develop  upon  such  gelatin  plate- 
cultures  are  seen  under  the  microscope  to  be  brownish- 
yellow  in  color,  spindle-shaped,  and  sharply  circum- 
scribed. When  superficial  they  are  larger  and  form  a 
bluish  iridescent  layer  with  notched  edges.  These  colo- 
nies are  often  described  as  resembling  grape-vine  leaves. 


TYPHOID  FEVER.  371 

The  center  of  the  superficial  colonies  is  the  only  portion 
which  shows  the  yellowish-brown  color.  The  margins 
of  the  colony  appear  somewhat  reticulated.  The  gelatin 
is  not  liquefied. 

Unfortunately,  the  appearances  of  the  colonies  of  the 
Bacillus  typhi  and  the  Bacillus  coli  communis  are  iden- 
tical, and  make  it  next  to  impossible  to  select  a  single 
colony  of  either  with  any  certainty.  The  only  solution 
of  the  problem  is  to  transfer  a  large  number  of  colonies 
to  some  culture-medium  in  which  a  characteristic  of  one 
or  the  other  species  is  manifested,  and  then  study  the 
growth ;  or  to  grow  the  colonies  upon  some  special 
medium  in  which  differences,  such  as  rapidity  of  growth 
or  acid-production,  etc.  cause  the  colonies  of  the  differ- 
ent species  to  assume  characteristic  appearances. 

A  method  recently  suggested  by  Eisner1  has  materially 
aided  the  separation  of  these  allied  bacteria  by  using  a 
culture-medium  upon  which  the  two  bacilli  develop  dif- 
ferently. 

The  Eisner  medium  can  be  made  by  allowing  i  kgm. 
of  grated  potatoes  (the  small  red  German  potato  is  best) 
to  macerate  in  i  liter  of  water  over  night.  The  juice  is 
carefully  pressed  out,  and  filtered  cold  to  get  rid  of  as 
much  starch  as  possible.  The  filtrate  is  now  boiled  and 
filtered  again.  The  next  step  is  a  neutralization,  in 
which  Eisner  used  litmus  as  an  indicator,  and  added  2.5- 
3  c.cm.  of  a  -^  normal  solution  of  sodium  hydrate  to  each 
10  c.cm.  of  the  juice.  Abbott  prefers  to  use  phenol- 
phthalein  as  an  indicator.  The  final  reaction  should  be 
slightly  acid.  Ten  per  cent,  of  gelatin  (no  peptone  or 
sodium  chlorid)  is  now  dissolved  in  the  solution,  which 
is  boiled  for  the  purpose,  and  must  then  be  again  neu- 
tralized to  the  same  point  as  before.  After  filtration,  the 
medium  receives  the  addition  of  i  per  cent,  of  potassium 
iodid.  It  is  filled  into  tubes  and  sterilized. 

When  water  or  feces  suspected  of  containing  the  tv- 
phoid  bacillus  are  mixed  in  this  medium  and  poured 

1  Zeitschrift  fur  Hygiene,  xxii.,  Heft  I,  1895;  Dec.  6,  1896. 


372  PATHOGENIC  BACTERIA. 

upon  plates,  no  bacteria  develop  well  except  the  colon 
bacillus  and  the  typhoid  bacillus. 

These  two  bacteria,  however,  differ  very  markedly  in 
their  appearance  upon  the  medium,  for  the  colon  bacillus 
appears  as  usual  in  twenty-four  hours,  while  at  that  time, 
if  present,  the  typhoid  bacillus  will  have  produced  no 
colonies  discoverable  by  the  microscope. 

It  is  only  after  forty-eight  hours,  long  after  the  colon 
colonies  have  attained  considerable  size  and  are  conspic- 
uous, that  the  little  colonies  of  the  typhoid  bacillus 
appear  as  small,  round,  shining,  dew-like  points,  which 
are  finely  granular  and  in  marked  contrast  to  their 
coarsely  granular  predecessors.  Unfortunately,  many  of 
the  small  colonies  that  develop  in  Eisner's  medium  sub- 
sequently prove  to  be  those  of  the  colon  bacillus. 

Kashida !  prefers  to  make  the  differential  diagnosis  by 
observing  the  marked  acid  production  of  the  Bacillus  coli 
upon  a  medium  consisting  of  bouillon  containing  ij^  per 
cent,  of  agar,  2  per  cent,  of  milk-sugar,  i.o  per  cent,  of 
urea,  and  30.0  per  cent,  of  tincture  of  litmus.  The  cul- 
ture-medium should  be  blue.  When  liquefied  and  inocu- 
lated with  the  colon  bacillus,  poured  into  Petri  dishes, 
and  stood  for  sixteen  to  eighteen  hours  in  the  incubator, 
the  blue  color  passes  off  and  the  culture-medium  becomes 
red.  If  a  glass  rod  dipped  in  hydrochloric  acid  be  held 
over  the  dish,  vapor  of  ammonium  chlorid  is  given  off. 
The  typhoid  bacillus  produces  no  acid  in  this  medium, 
and  there  is  consequently  no  change  in  its  color. 

For  the  differentiation  of  the  typhoid  bacillus  from  the 
allied  bacillary  forms,  Hiss2  recommends  the  use  of  two 
special  media.  The  first  consists  of  5  grams  of  agar-agar, 
80  grams  of  gelatin,  5  grams  of  Liebig's  beef-extract,  5 
grains  of  sodium  chlorid,  and  10  grams  of  glucose  to  the 
liter.  The  agar  is  dissolved  in  the  1000  c.cm.  of  water,  to 
which  have  been  added  the  beef-extract  and  sodium  chlorid. 
When  the  agar  is  completely  melted  the  gelatin  is  added 

1  Cfntralbl.  f.  Bakt.  it.  /\/;/V/V;/£.,  Bd.  xxi.,  Nos.  2O  and  21,  June  24,  1897. 
J  Journal  of  Experimental  Medicine,  Nov.,  1897,  vol.  ii.,  No.  6. 


TYPHOID  FEVER.  373 

and  thoroughly  dissolved  by  a  few  minutes'  boiling.  The 
medium  is  then  titrated  to  determine  its  reaction,  phenol- 
phthalein  being  used  as  the  indicator,  and  enough  HC1  or 
NaOH  added  to  bring  it  to  the  desired  reaction — i.  e.  a  re- 
action indicating  i.  5  per  cent,  of  normal  acid.  To  the  clear 
medium  add  one  or  two  eggs,  well  beaten  in  25  c.cm.  of 
water;  boil  for  forty-five  minutes,  and  filter  through  a 
thin  filter  of  absorbent  cotton.  Add  the  glucose  after 
cleaning. 

The  medium  is  used  in  tubes,  in  which  it  is  planted  by 
the  ordinary  puncture.  The  typhoid  bacillus  alone,  of 
many  of  the  allied  forms  studied,  has  the  power  of  cloud- 
ing this  medium  uniformly  without  showing  streaks  or 
gas-bubbles. 

The  second  medium  is  used  for  plating.  It  contains 
10  grams  of  agar,  25  grams  of  gelatin,  5  grams  of  beef- 
extract,  5  grams  of  sodium  chlorid,  and  10  grams  of  glu- 
cose. The  method  of  preparation  is  the  same  as  for  the 
tube-medium,  care  always  being  taken  to  add  the  gela- 
tin after  the  agar  is  thoroughly  melted,  so  as  not  to  alter 
this  ingredient  by  prolonged  exposure  to  high  tempera- 
ture. This  preparation  should  never  contain  less  than  2 
per  cent,  of  normal  acid.  Of  all  the  organisms  with 
which  Hiss  experimented,  the  Bacillus  typhosus  alone 
displayed  the  power  of  producing  thread-forming  colonies 
upon  this  medium. 

The  colonies  of  the  typhoid  bacillus  when  deep  in  the 
medium  appear  small,  generally  spherical,  with  a  rough, 
irregular  outline,  and  by  transmitted  light  are  of  a  vitreous 
greenish  or  yellowish-green  color.  The  most  character- 
istic feature  consists  of  well-defined  filamentous  out- 
growths, ranging  from  a  single  thread  to  a  complete 
fringe  about  the  colony.  The  young  colonies  are,  at 
times,  composed  solely  of  threads.  The  fringing  threads 
generally  grow  out  nearly  at  right  angles  to  the  periphery 
of  the  colony. 

The  colonies  of  the  colon  bacillus  are,  on  the  average, 
larger  than  those  of  the  typhoid  bacillus;  they  are  spher- 


374  PATHOGENIC  BACTERIA. 

ical  or  of  a  whetstone  form,  and  by  transmitted  light  are 
darker,  more  opaque,  and  less  refractive  than  the  typhoid 
colonies.  By  reflected  light,  to  the  unaided  eye  they  are 
pale  yellow.  The  surface-colonies  are  large,  round,  irreg- 
ularly spreading,  and  are  brown  or  yellowish-brown  in 
color.  Hiss  claims  that  by  the  use  of  these  reagents  the 
typhoid  bacillus  can  be  readily  detected  in  typhoid  stools. 

When  transferred  to  gelatin  puncture-cultures  the  ba- 
cilli develop  along  the  entire  track  of  the  wire,  with  the 
formation  of  minute  confluent  spherical  colonies.  A 
small  thin  whitish  layer  develops  upon  the  surface  near 
the  center.  The  gelatin  is  not  liquefied,  but  sometimes 
is  slightly  clouded  in  the  neighborhood  of  the  growth. 
The  growth  upon  the  surface  of  obliquely  solidified  gela- 
tin, agar-agar,  or  blood-serum  is  not  very  luxuriant.  It 
forms  a  thin,  moist,  translucent,  non-characteristic  band 
with  smooth  edges. 

Upon  potato  a  growth  formerly  regarded  as  character- 
istic takes  place.  When  the  potato  is  inoculated  and 
stood  in  the  incubating-oven,  no  growth  can  be  detected 
at  the  end  of  the  second  day,  unless  the  observer  be 
skilled  and  the  examination  thorough.  If,  however,  the 
medium  be  touched  with  a  platinum  wire,  it  is  discovered 
that  its  entire  surface  is  covered  with  a  rather  thick,  in- 
visible layer  of  a  sticky  vegetation  which  the  microscope 
shows  to  be  made  up  of  bacilli.  No  other  bacillus  gives 
the  same  kind  of  growth  upon  potato.  Unfortunately,  it 
is  not  constant,  for  occasionally  there  will  be  encountered 
a  typhoid  bacillus  which  will  show  a  distinct  yellowish 
or  brownish  color.  The  typical  growth  seems  to  take 
place  only  when  the  reaction  of  the  potato  is  acid. 

In  bouillon  the  only  change  produced  by  the  growth  of 
the  bacillus  is  a  diffuse  cloudiness. 

In  milk  a  slight  and  slow  acidity  is  produced.  The 
growth  in  milk  is  not  accompanied  by  coagulation. 

The  chief  hindrance  to  the  ready  isolation  of  the 
typhoid  bacillus  is  the  closely-allied  Bacillus  coli  com- 
iniiiiis.  This  organism,  being  habitually  present  in  the 


TYPHOID  FEVER.  375 

intestine,  exists  there  in  typhoid  fever,  and  adds  no  little 
complication  to  the  bacteriological  diagnosis  by  respond- 
ing in  exactly  the  same  manner  as  the  typhoid  bacillus 
to  the  action  of  carbolic  acid,  by  having  colonies  almost 
exactly  like  those  of  typhoid,  by  growing  in  exactly  the 
same  manner  upon  gelatin,  agar-agar,  and  blood-serum, 
by  clouding  bouillon  in  the  same  way,  by  being  of  almost 
exactly  the  same  shape  and  size,  by  having  flagella,  by 
being  motile,  and,  in  fact,  by  so  many  pronounced  simi- 
larities as  almost  to  warrant  the  assertion  of  some  that  it 
and  the  typhoid  bacillus  are  identical. 

Not  the  least  significant  fact  about  the  colon  bacillus 
is  that  it  is  also  pathogenic  and  capable  of  exciting  acute 
inflammatory  processes  which  are  not  infrequent,  and 
which  sometimes  serve  to  increase  the  seriousness  of 
typhoid  fever. 

At  the  present  time  we  are  in  more  or  less  of  a  quan- 
dary about  this  extraordinary  resemblance,  but  base  our 
differentiation  of  the  species  upon  certain  constant,  slight, 
but  distinct  differences. 

The  typhoid  bacillus  does  not  produce  indol. 

The  open  lymphatics  and  vessels  of  the  intestinal  ulcers 
of  typhoid  favor  the  absorption  of  the  bacteria  in  the  diges- 
tive tract,  and  the  colon  bacillus  enters  the  blood  no 
longer  to  be  a  saprophyte,  but  now  to  be  a  virulent  pus- 
producer,  and  in  many  cases  of  typhoid  we  find  suppura- 
tions and  other  milder  inflammations  due  to  this  microbe. 
This  is  also  a  stumbling-block,  for  the  typhoid  bacillus 
when  distributed  through  the  blood  may  act  in  exactly 
the  same  manner. 

The  typhoid  bacillus  may  enter  the  body,  at  times, 
through  dust  (Klemperer  and  Levy),  but  no  doubt,  in  the 
great  majority  of  cases,  enters  the  digestive  tract  at  once 
through  the  mouth.  It  may  possibly  enter  through  the 
rectum  at  times,  as  illustrated  by  the  mention  which 
Eichhorst  makes  of  the  infection  of  soldiers  in  military 
barracks  through  the  wearing  of  drawers  previously  worn 
by  comrades  who  had  suffered  from  typhoid. 


376 


PATHOGENIC  BACTERIA. 


When  ingested  the  resisting  power  of  the  bacillus  per- 
mits it  to  pass  uninjured  through  the  acid  secretions  of 
the  stomach  and  to  enter  the  intestine,  where  the  chief 
local  disturbances  are  set  up. 

The  bacilli  enter  the  solitary  glands  and  Peyer's  patches, 
and  multiply  slowly  during  the  one  to  three  weeks  of  the 
incubation  of  the  disease.  The  immediate  result  of  their 
residence  in  these  lymphatic  structures  is  increase  in  the 
number  of  cells,  and  ultimately  the  necrosis  and  slotigh- 


FIG.  108.— Intestinal  perforation  in  typhoid  fever.  Observe  the  threads  of 
tissue  obstructing  the  opening.  (Museum  of  the  Pennsylvania  Hospital.) 
(Keen,  Surgical  Complications  and  Sequels  of  Tvphoid  Fever.} 

ing  which  cause  the  typical  post-mortem  lesion  (Fig.  108). 
From  the  intestinal  lymphatics  the  bacilli  pass,  in  all 
probability,  to  the  mesenteric  glands,  which  become  en- 
larged and  softened,  and  finally  extend  to  the  spleen  and 
liver,  and  sometimes  to  the  kidneys.  The  growth  of  the 
bacilli  in  the  kidneys  causes  the  albuminuria  of  the  dis- 
ease. Sometimes  under  these  conditions  the  bacilli  may 


TYPHOID  FEVER.  377 

be  found  in  the  urine.  P.  Horton  Smith  l  found  the  ba- 
cilli in  the  urine  in  three  out  of  seven  cases  which  he 
investigated.  They  did  not  occur  before  the  third  week, 
and  remained  in  one  case  twenty-two  days  after  cessation 
of  the  fever.  Sometimes  they  were  present  in  immense 
numbers.  Their  occurrence,  no  doubt,  depends  upon 
their  growth  in  the  kidney  and  descent  with  the  urine. 
It  is  of  importance  from  a  sanitary  point  of  view  to 
remember  that  the  urine  as  well  as  the  feces  is  infec- 
tious. Occasionally  the  bacilli  succeed  in  entering  the 
general  circulation,  and,  finding  a  lodgement  at  some 
remote  part  of  the  body,  set  up  local  inflammatory  pro- 
cesses sometimes  terminating  in  suppuration. 

Weichselbaum  has  seen  general  peritonitis  from  rup- 
ture of  the  spleen  in  typhoid  fever  with  escape  of  the 
bacilli.  Ostitis,  periostitis,  and  osteomyelitis  are  very 
common  results  of  the  lodgement  of  the  bacilli  in  bony 
tissue,  and  Ohlmacher  has  found  the  bacilli  in  suppura- 
tions of  the  membranes  of  the  brain.  The  bacilli  are 
also  encountered  in  other  local  suppurations  occurring 
in  or  following  typhoid  fever.  Flexner  and  Harris2  have 
seen  a  case  in  which  the  distribution  of  the  bacilli  was 
sufficiently  widespread  to  constitute  a  real  septicemia, 
the  bacillus  being  isolated  from  various  organs  of  the 
body,  and  shown  to  be  the  true  bacillus  of  Eberth  by 
all  the  specific  laboratory  tests,  but  in  which  there  were 
no  intestinal  lesions. 

The  bacilli  can  be  found  in  the  intestinal  lesions,  in 
the  mesenteric  glands,  in  the  spleen,  in  the  liver,  in  the 
kidneys,  and  in  any  local  lesions  which  may  be  present. 
Their  scattered  distribution  and  their  occurrence  in 
minute  clumps  have  already  been  alluded  to.  They 
should  always  be  sought  for  at  first  with  a  low  power 
of  the  microscope. 

Ordinarily  no  bacilli  can  be  found  in  the  blood,  but 
it  has  been  shown  that  the  blood  in  the  roseolae  some- 

1  Brit.  Med.  Jour.,  Feb.  13,  1897. 

2  Bull,  of  the  Joints  Hopkins  Hospital,  Dec  ,  1897. 


378  PATHOGENIC  BACTERIA. 

times  contains  them,  so  that  the  eruption  may  be  regarded 
as  one  of  the  local  irritative  manifestations  of  the  bacillus. 

The  amount  of  local  disturbance,  in  proportion  to  the 
constitutional  disturbance,  is,  in  the  majority  of  cases, 
slight,  and  almost  always  partakes  of  a  necrotic  charac- 
ter, which  suggests  that  in  typhoid  we  have  to  do  with  a 
toxic  bacterium  whose  disease-producing  capacity  resides 
in  the  elaboration  of  a  toxic  substance.  This,  indeed, 
is  true,  for  Brieger  and  Frankel  have  separated  from 
bouillon  cultures  a  toxalbumin  which  they  thought  to  be 
the  specific  poison.  Klemperer  and  Levy  also  point  out 
further  clinical  proof  in  certain  exceptional  cases  dying 
with  the  typical  picture  of  typhoid,  yet  without  char- 
acteristic post-mortem  lesions,  the  only  confirmation  of 
the  diagnosis  being  the  discovery  of  the  bacilli  in  the 
spleen. 

Pfeiffer  and  Kolle  found  that  the  toxic  substance  resided 
only  in  the  bodies  of  the  bacilli,  and  could  not,  like  the 
toxins  of  diphtheria  and  tetanus,  be  dissolved  in  the  cul- 
ture-medium. This  was  an  obstacle  to  their  immuniza- 
tion-experiments as  well  as  those  of  Loftier  and  Abel, 
later  to  be  described,  for  the  only  method  of  immuniz- 
ing animals  to  large  quantities  of  the  bacilli  was  to  make 
massive  agar-agar  cultures,  scrape  the  bacilli  from  the 
surface,  and  distribute  them  through  nutrient  bouillon. 

When  injected  into  guinea-pigs  the  typhotoxin  of 
Brieger  is  productive  of  increased  secretion  of  saliva,  in- 
creased rapidity  of  respiration,  diarrhea,  and  mydriasis, 
and  usually  causes  a  fatal  termination  in  from  twenty- 
four  to  forty-eight  hours. 

As  the  discovery  of  the  bacilli  in  the  spleen,  and  espe- 
cially the  securing  of  a  pure  culture  of  the  bacilli  from 
the  spleen,  are  sometimes  attended  with  considerable  dif- 
ficulty because  of  the  dissemination  of  the  colonies 
throughout  the  organ,  E.  Frankel  recommends  that  as 
soon  as  the  organ  is  removed  from  the  body  it  be  wrapped 
in  cloths  wet  with  a  solution  of  bichlorid  of  mercury  and 
kept  for  three  days  in  a  warm  room,  in  order  that  a  con- 


TYPHOID  FEVER.  379 

siderable  and  massive  development  of  the  bacilli  may 
take  place. 

Typhoid  fever  is  a  disease  which  is  communicable  to 
animals  with  difficulty.  They  are  not  affected  by  bacilli 
in  fecal  matter  or  in  pure  culture  mixed  with  the  food, 
and  are  not  diseased  by  the  injection  into  them  of  blood 
from  typhoid  patients.  Gaffky  failed  completely  to  pro- 
duce any  symptoms  suggestive  of  typhoid  fever  in  rab- 
bits, guinea-pigs,  white  rats,  mice,  pigeons,  chickens, 
and  calves,  and  found  that  Java  apes  could  feed  daily 
upon  food  polluted  with  typhoid  germs  for  a  considerable 
time,  yet  without  symptoms.  The  introduction  of  pure 
cultures  into  the  abdominal  cavity  of  most  animals  is 
without  effect.  Frankel  and  Simon  found  that  when 
pure  cultures  were  injected  into  mice,  rabbits,  and  guinea- 
pigs  the  animals  died. 

Germano  and  Maurea  found  that  mice  succumbed  in 
from  one  to  three  days  after  intraperitoneal  injection  of 
1-2  c.  cm.  of  a  twenty-four-hour-old  bouillon  culture.  Sub- 
cutaneous injections  in  rabbits  and  dogs  caused  abscesses. 

Losener  found  the  introduction  of  3  mgr.  of  an  agar- 
agar  culture  into  the  abdominal  cavity  of  guinea-pigs  to 
be  fatal. 

When  animals  are  treated  in  the  manner  described  in 
the  chapter  upon  Cholera — /.  «?.  the  gastric  contents  ren- 
dered alkaline,  a  large  quantity  of  laudanum  injected 
into  the  peritoneal  cavity,  and  the  bacilli  introduced 
through  an  esophageal  catheter — Klemperer,  Levy,  and 
others  found  that  there  was  produced  an  intestinal  con- 
dition which  very  much  resembled  typhoid  as  it  occurs  in 
man.  The  virulence  of  the  bacillus  can  be  very  greatly 
increased  by  rapid  passage  from  guinea-pig  to  guinea-pig. 

In  the  experiments  of  Chantemesse  and  Widal  the 
symptoms  following  the  injection  of  virulent  culture  into 
guinea-pigs  were  briefly  as  follows:  "Very  shortly  after 
the  inoculation  there  is  a  rise  of  temperature,  which 
continues  from  one  to  four  hours,  and  is  succeeded  by  a 
depression  of  the  temperature,  which  continues  to  the 


380  PATHOGENIC  BACTERIA. 

fatal  issue.  Meteorism  and  great  tenderness  of  the  abdo- 
men are  observed.  At  the  autopsy  a  sero-fibrinous  or 
sero-purulent  peritonitis  is  observed — sometimes  hemor- 
rhagic.  There  is  also  generally  a  pleurisy,  either  serous 
or  hemorrhagic.  All  the  abdominal  viscera  are  con- 
gested. The  intestine  is  congested — contains  an  abun- 
dant mucous  secretion.  The  Peyer  patches  are  enlarged. 
The  spleen  is  enlarged,  blackish,  and  often  hemorrhagic. 
In  cases  which  are  prolonged  the  liver  is  discolored.  The 
kidneys  are  congested,  the  adrenals  filled  with  blood. 

"  In  such  cases  the  bacillus  can  be  found  upon  the  in- 
flamed serous  membranes,  in  the  inflammatory  exudates, 
in  the  spleen  in  large  numbers,  in  the  adrenals,  the  liver, 
the  kidneys,  and  sometimes  in  the  lungs.  The  blood  is 
also  infected,  but  to  a  rather  less  degree. 

"In  cases  described  as  chronic,  the  bacillus  disappears 
completely  in  from  five  to  twenty-four  hours,  and  pro- 
duces but  one  lesion,  a  small  abscess  at  the  point  of  inoc- 
ulation. 

u  Sanarelli  has  observed  that  if  some  of  the  poisonous 
products  of  the  colon  bacillus  or  the  Proteus  vulgaris  be 
injected  into  the  abdominal  cavity  of  an  animal  recover- 
ing from  a  chronic  case,  it  speedily  succumbs  to  typical 
typhoid  fever." 

Petruschky1  found  that  mice  that  recovered  from  sub- 
cutaneous injections  of  typhoid  cultures  frequently  suf- 
fered from  a  more  or  less  widespread  necrosis  of  the  skin 
at  the  point  of  injection. 

I  experienced  great  difficulty  in  immunizing  a  horse  to 
the  disease,  because  every  injection  of  virulent  living 
organisms  was  followed  by  a  necrosis  equalling  in  size  the 
distended  area  of  subcutaneous  tissue. 

Large  quantities  of  filtered  cultures  produce  symp- 
toms similar  to  those  resulting  from  inoculation  with 
the  bacilli.  The  toxic  product  of  the  bacilli  is,  how- 
ever, practically  insoluble,  and,  according  to  the  ex- 
periments of  Loffler  and  Abel  and  those  of  Pfeiffer  and 

1   '/.fitschrift fur  Hygiene,  Bd.  xii.,  1892,  p.  261. 


TYPHOID  FEVER.  381 

Kolle,  cannot  be  separated  from  the  bodies  of  the  bacilli 
producing  it. 

Animals  can  easily  be  immunized  to  this  bacillus,  and 
then,  according  to  Chantemesse  and  Widal,  develop  in 
their  blood  an  antitoxic  substance  capable  of  protecting 
other  animals.  Stern  L  has  also  found  that  in  the  blood 
of  human  convalescents  a  substance  exists  which  has  a 
protective  effect  upon  guinea-pigs.  His  observation  is  in 
accordance  with  a  previous  one  by  Chantemesse  and 
Widal,  and  has  recently  been  abundantly  confirmed. 

The  immunization  of  dogs  and  goats  by  the  introduc- 
tion of  increasing  doses  of  virulent  cultures  has  been 
achieved  by  Pfeiffer  and  Kolle2  and  by  LofBer  and  Abel.3 
From  these  animals  serums  were  secured  not  exactly  an- 
titoxic, but  anti-infectious  or  auti-microbic  in  operation, 
and  possessed  of  marked  specific  germicidal  action  upon 
the  typhoid  bacilli  when  simultaneously  introduced  into 
the  peritoneal  cavity  of  guinea-pigs. 

The  action  of  the  typhoid  serum  is  specific,  and  exerts 
exactly  the  same  action  upon  the  typhoid  bacilli  as  the 
cholera  serum  exerts  upon  the  cholera  spirilla,  killing 
and  dissolving  them  (Pfeiffer' s  phenomenon). 

So  far,  no  serum  has  been  produced  that  is  efficacious 
in  human  medicine. 

The  specific  reaction  of  the  serum  can  be  used  to  dif- 
ferentiate cultures  of  the  colon  and  typhoid  bacilli,  the 
typhoid  bacilli  alone  exhibiting  the  specific  effect  of  the 
typhoid  serum. 

Christophers4  found  that  the  serum  from  typhoid 
patients  occasionally  caused  agglutinations  in  cultures 
of  the  colon  bacillus,  but  concludes  that  this  does  not 
lessen  the  specificity  of  the  reaction,  as  there  may  be 
two  combined  specific  actions  of  these  serums.  Experi- 
ments on  rabbits  established  that  typhoid  and  colon 
serums  could  be  produced,  each  specific  in  its  agglutin- 

1  Zeitschrift  fur  Hygiene,  xvi.,  1894,  p.  458.  2  Ibid.,  1896. 

3  Centralbl.f.  Bakt.  u.  Parasitenk.,  Bd.  xix.,  No.  23,  p.  51,  Jan.  23, 1896. 
*  Brit.  Med.  Jour.,  Jan.  8,  1898. 


382  PATHOGENIC  BACTERIA. 

ating  power  upon  bouillon  cultures  of  its  respective 
organism. 

Loffler  and  Abel  also  prepared  a  colon  serum  which 
exerted  a  like  specific  action  upon  the  colon  bacillus, 
but  was  without  effect  upon  the  typhoid  bacillus. 

The  serum  of  immunized  animals  has  been  found  to 
destroy  the  motility  of  the  typhoid  bacilli  in  a  few  mo- 
ments, and  to  cause  them  to  group  together.  Widal 
found  that  the  serum  of  convalescents  and  of  individuals 
suffering  from  the  acute  disease  possessed  the  same  power, 
and  suggested  that  this  specific  action  might  prove  a  val- 
uable adjunct  in  diagnosis. 

Wyatt  Johnston 1  and  McTaggert  worked  upon  the 
subject,  and  found  that  a  drop  of  blood  from  a  typhoid 
patient,  dried  upon  paper  and  kept  for  some  time,  when 
moistened  and  brought  in  contact  with  a  culture  of  the 
bacilli  was  still  potent  to  bring  about  a  characteristic 
effect.  When  such  a  preparation  in  the  u  hanging  drop  n 
is  watched  under  the  microscope  the  typhoid  bacilli  are 
found  to  be  paralyzed  •  in  from  one  minute  to  half  an 
hour,  and  subsequently  to  collect  in  masses — agglutina- 
tions. This  reaction  may  occasionally  be  brought  about 
by  normal  blood  if  insufficiently  diluted,  but  is  charac- 
teristic enough  to  be  very  useful  in  the  diagnosis  of  ob- 
scure cases.  In  a  later  paper  Johnston  states  that  to  ob- 
tain a  satisfactory  reaction  an  attenuated  typhoid  bacillus 
is  more  useful  than  a  highly  virulent  one. 

My  own  experiments  have  satisfied  me  of  the  value  of 
the  test,  both  for  making  a  diagnosis  of  the  disease  and 
for  confirming  the  species  of  the  bacillus  in  doubtful 
cases. 

It  is  now  the  opinion  of  all  observers  that  cessation  of 
motion  and  agglutination  of  the  bacteria,  resulting  from 
the  contact  of  typhoid  bacilli  and  typhoid  serum,  are 
inconclusive  for  diagnostic  purposes  unless  the  reaction 
follows  the  combination  of  a  suitable  culture  and  a 
deft  n  ite  y  u  a  u  tity  of  sent  ni . 

1  Montreal  Mea.  Journal ',  March,  1897. 


TYPHOID  FEVER.  383 

The  thorough  investigations  of  Wyatt  Johnston  and 
his  associates  in  Montreal  have  shown  that  reliable  reac- 
tions can  only  be  secured  when  the  cultures  employed  are 
of  an  ordinarily  virulent  typhoid  bacillus,  and  are  grown 
in  an  alkaline  medium  for  about  twenty-four  hours. 

I  prefer  fresh  agar-agar  cultures,  distributed  throughout 
sterile  clean  water,  rather  than  bouillon  cultures,  because 
of  the  larger  number  of  bacteria  in  the  former,  the  con- 
sequently greater  number  of  agglutinations  formed,  and 
the  readiness  with  which  they  are  found  upon  micro- 
scopic examination.  It  is  necessary,  however,  to  make 
a  microscopic  examination  of  the  diluted  culture  before 
adding  the  serum  or  blood,  in  order  to  be  sure  that  there 
are  no  natural  clumps  of  bacteria  present  to  simulate  the 
specific  agglutinations.  This  is  of  great  importance. 
The  natural  clumps  of  bacilli  are  more  apt  to  occur  in 
cultures  grown  upon  fresh,  moist  agar-agar  than  upon 
that  kept  for  a  short  time  until  the  surface  has  become 
partially  dried.  The  chief  difficulty  experienced  in 
making  the  test  seems,  at  present,  to  reside  in  the  prepa- 
ration of  the  blood  in  accurate  dilution — i.  e.  securing 
it  in  measured  amounts. 

The  original  method  of  Widal,  to  collect  about  5  c.cin. 
of  blood  in  a  test-tube  by  the  introduction  of  a  hypo- 
dermic needle  into  a  vein,  is  a  rather  more  serious  and 
disturbing  operation  than  most  patients  care  to  undergo 
for  purposes  of  diagnosis. 

Blood  dried  upon  paper,  as  suggested  by  Johnston,  or 
upon  glass,  while  extremely  convenient  for  transporta- 
tion, is  not  susceptible  of  accurate  dilution  for  quantita- 
tive estimation. 

Cabot  has  successfully  made  dilutions  with  a  medicine- 
dropper,  by  using  one  drop  of  blood  and  as  many  drops 
of  culture,  dropped  from  the  same  instrument,  as  were 
necessary  for  the  desired  dilution.  This  method  seems 
to  be  very  practical,  but  can  only  be  employed  at  the 
bedside,  or  where  it  is  not  necessary  to  keep  or  trans- 
port the  blood. 


384  PATHOGENIC  BACTERIA. 

In  the  absence  of  a  satisfactory  method  of  securing 
definite  small  quantities  of  blood  for  immediate  or  sub- 
sequent use,  I  was  led  to  make  some  experiments  with 
capillary  tubes  to  determine  their  possible  value  for  the 
purpose. 

It  is  a  well-known  physical  phenomenon  that  in  clean 
capillary  tubes  fluids  are  attracted  to  a  height  varying 
according  to  the  diameter  of  the  tube  and  the  density  of 
the  fluid.  In  tubes  of  equal  diameter  the  height  of  the 
column  is  invariably  the  same. 

Such  tubes  can  be  made  by  heating  a  piece  of  ordinary 
glass  tubing,  such  as  is  to  be  found  in  every  laboratory, 
in  a  Bunsen  flame  for  a  few  minutes  until  it  becomes  red 
and  soft,  removing  the  glass  from  the  flame,  and  then 
pulling  upon  the  ends  steadily  and  slowly  until  the  tube 
is  drawn  out  to  the  desired  diameter.  The  errors  to  be 
avoided  in  making  the  tubes  will  be — heating  too  much 
and  making  the  glass  too  soft,  drawing  out  the  tube 
while  still  in  the  flame,  and  drawing  too  rapidly.  The 
result  of  these  erroneous  methods  will  be  that  the  tubes 
are  much  shorter  and  finer  than  is  desired.  A  few  mo- 
ments' practice  will  show  just  how  the  manipulation 
should  be  done  to  secure  the  best  results. 

The  fact  was,  however,  established  that  tubes  of  about 
the  same  diameter  showed  almost  no  variation  in  the 
quantity  of  liquid  contained.  So  little  was  the  difference 
in  the  length  of  the  column  and  the  weight  of  the  con- 
tained blood  in  tubes  recognized  by  the  eye  to  have  uni- 
form caliber  that  I  have  no  hesitation  in  recommend- 
ing an  application  of  the  capillary  tube  for  securing 
small  measured  quantities  of  blood  for  the  specific  typhoid 
tests  and  similar  experiments. 

The  application  of  the  method  is  simple  and  consists  in: 

1.  Accurately   weighing    the    amount   of   blood    that 
enters  a  capillary  tube  of  a  size  arbitrarily  selected  as  a 
standard. 

2.  The  manufacture  of  a  large  number  of  tubes  of  the 
same  size. 


TYPHOID  FEVER.  385 

3.  The  dilution  of  the  known  quantity  of  blood  con- 
tained in  the  tube  with  a  measured  quantity  of  the 
bouillon,  or  diluted  agar-agar,  culture  of  the  bacillus. 

The  standard  tube  that  I  adopted  had  a  diameter 
about  equal  to  the  K  string  of  a  violin.  A  larger  or 
smaller  tube  would  have  done  quite  as  well.  In  such  a 
tube  the  column  of  blood  rises  about  an  inch  and  weighs 
about  0.018  gram.  As  personal  equation  in  judging 
size  is  a  marked  source  of  error,  the  experimenter  must 
work  out  his  own  standard  tube  and  not  adopt  that  which 
has  just  been  given.  It  is  important  to  know  the  length 
of  the  column  that  has  a  certain  weight,  because,  as  each 
tube  is  not  separately  measured  and  graduated,  the  two 
chief  means  of  avoiding  error  will  be  (i)  to  have  the 
tubes  as  nearly  as  possible  of  equal  diameter,  and  (2)  to 
prove  them  to  be  so  by  observing  that  the  columns  of 
fluid  they  contain  when  used  are  of  the  same  length,  re- 
jecting one  after  another  all  the  tubes  which  seem  to 
the  eye  to  have  the  proper  caliber,  but  in  which  the 
column  is  obviously  longer  or  shorter  than  that  of  the 
original  tube. 

Keeping  the  standard  tube  before  him  as  a  guide,  and 
using  a  Bunsen  flame — which  is  better  than  a  blowpipe, 
because  it  does  not  heat  the  glass  so  rapidly  and  make  it 
so  soft — the  experimenter  prepares  one  hundred  or  more 
capillary  tubes  as  nearly  as  possible  of  the  same  size  as 
that  of  the  standardized  tube.  All  the  irregular  sizes 
are  rejected,  and  the  suitable  sizes  cut  into  portions 
about  three  inches  long.  These  pieces,  which  should 
number  several  hundred  (it  is  economy  to  make  a  large 
number  at  a  time),  are  now  carefully  sorted,  being  com- 
pared with  the  standard  tube  at  both  ends,  and  thrown 
away  if  too  large  or  too  small  at  either.  It  is  best  to 
sort  the  tubes  twice  on  different  days,  or  have  several 
different  persons  go  over  them  all.  Of  course,  some 
tubes  of  quite  different  caliber  will,  in  spite  of  all  pre- 
cautions, remain  in  the  bundle,  but  this  is  no  serious 
matter,  because  at  the  last  moment  the  height  of  the 

25 


386  PATHOGENIC  BACTERIA. 

column  to  which  the  blood  rises  can  be  taken  as  a  proof 
of  actual  variation.  It  may  be  true  that  no  two  of  the 
tubes  have  exactly — absolutely — the  same  contents,  but 
when  the  given  precautions  are  taken  the  variation  will 
be  so  small  as  to  make  no  significant  error  in  the  results 
obtained. 

The  use  of  the  tubes  is  extremely  simple.  The  ordi- 
nary puncture  is  made  in  the  lobule  of  the  ear  or  the  fin- 
ger-tip of  the  patient,  and  one  end  of  one  of  the  tubes 
touched  to  the  surface  of  the  oozing  drop  and  held  there 
until  the  blood  ceases' to  rise  in  the  tube.  So  little  blood 
is  required  that  a  number  of  tubes  may  be  filled  with  the 
blood  from  a  single  puncture  if  desired.  The  blood  in 
the  tube  coagulates  in  a  few  minutes,  and  can  be  allowed 
to  dry,  or  be  drawn  to  the  central  portion  of  the  tube  and 
sealed  in  by  fusing  the  ends  in  a  flame  if  it  be  desired  to 
keep  it  moist. 

When  the  agglutination  reaction  is  to  be  made  the 
blood  should  not  be  blown  out  of  the  tube,  as  the  total 
quantity  contained  is  small  and  a  large  relative  quantity 
will  remain  in  the  tube.  A  better  method  is  to  crush  the 
tube  in  a  small  crucible  or  other  diminutive  vessel  and 
dissolve  its  contents  directly  in  the  culture. 

The  proper  proportionate  amount  of  culture  is  meas- 
ured with  a  finely  graduated  pipette  (graduated  to  thous- 
andths of  a  cubic  centimeter),  the  calculation  according 
to  the  standard  tube  of  the  writer's  experiments  being: 
dilution  i  :  10  =  0.153  c.cm.  of  the  culture;  dilution 
i  :  100=  1.53  c.cm.  of  the  culture;  dilution  i  :  1000  = 
15.3  c.cm.  of  the  culture. 

The  now  recognized  specific  reaction  is  supposed  to 
take  place  in  dilutions  of  i  :  50,  which  would  require 
0.71  +  c.cm.  of  the  bouillon  or  diluted  agar  culture. 

The  culture  is  measured  into  the  little  crucible,  the 
blood-containing  portion  of  the  capillary  tube  broken  off, 
dropped  in,  and  subsequently  crushed  to  minute  frag- 
ments and  stirred  about  with  a  clean,  rounded,  glass  rod, 
and  a  drop  of  the  mixture  placed  as  a  "hanging  drop'* 


TYPHOID  FEVER.  387 

upon  the  stage  of  a  microscope  and  examined  for  the 
agglutinations. 

As  recent  extended  observations  have  shown  that  occa- 
sionally the  blood  of  healthy  men  and  animals  has  the 
power  of  producing  the  agglutinations,  the  consensus  of 
opinion  now  seems  to  be  in  favor  of  the  view  that  a  cer- 
tain dilution  of  the  blood  is  required  for  a  satisfactory 
diagnosis,  and  that  all  reactions  with  concentrations 
greater  than  one  part  of  blood  in  fifty  of  culture  may  be 
questionable,  while  less  concentrated  dilutions  are  almost 
positively  diagnostic.  A  time-limit  must  be  placed  upon 
the  experiment.  For  the  weak  dilution  not  more  than 
two  hours  should  be  required  for  a  perfect  reaction,  and 
for  the  stronger  solution  correspondingly  less  time  should 
be  required. 

A  curious  fact  that  should  not  be  overlooked  is  that  the 
agglutinating  substance  is  not  constantly  present  in  the 
blood,  but  sometimes  alternates,  being  present  for  several 
days  and  then  absent  for  a  day  or  two. 

The  agglutinating  power  of  the  blood  occurs  early  in 
the  course  of  typhoid,  and  in  typical  cases  seems  to  be 
present  in  the  first  week  of  actual  illness. 

A  point  that  should  not  be  forgotten  is  that  the  agglu- 
tination of  the  bacilli  seems  to  be  a  phenomenon  quite  in- 
dependent of  any  immunity  possessed  by  the  individual, 
and  therefore  is  not  an  "immunity-reaction."  Just  what 
the  agglutinating  substance  is,  has  not  yet  been  determined. 

The  agglutinations  are  occasionally  caused  by  the 
serum  and  dried  blood  from  other  diseases  than  typhoid, 
but  in  a  collection  of  4000  cases  it  was  shown  that  the 
errors  from  this  source  were  only  about  5  per  cent. 

Malvoz  l  has  experimented  with  a  number  of  chemicals, 
and  has  found  that  formaldehyd,  corrosive  sublimate,  per- 
oxid  of  hydrogen,  strong  alcohol,  and  anilin  colors  (such 
as  chrysoidin,  vesuvin,  and  safranin)  have  the  power  to 
produce  the  typical  agglutinations  even  in  very  dilute 
solutions. 

1  Ann.  de  T Inst.  Pasteur,  xl.,  7,  1897. 


388  PA  THOGENIC  BA  CTERIA. 

Wright  and  Semple  assert  that  dead  cultures  of  the 
typhoid  bacillus  may  be  used  for  the  test,  as  bacilli  killed 
by  a  temperature  of  60°  C.  agglutinate  perfectly.  They 
have  the  advantage  of  being  easily  kept. 

Rumpf,1  and  Kraus  and  Buswell2  report  a  number  of 
cases  of  typhoid  which  were  favorably  influenced  by  the 
introduction  hypodermically  of  small  quantities  of  steril- 
ized cultures  of  Bacillus  pyocyaneus.  These  experiments 
are  still  too  new  to  deserve  extended  mention. 

Following  the  lines  of  experimentation  suggested  by 
Haffkine's  researches  upon  preventive  vaccination  against 
cholera  Asiatica,  Pfeiffer  and  Kolle,  and  Wright  and  Sem- 
ple have  used  the  subcutaneous  injection  of  sterilized  cul- 
tures as  a  prophylactic  measure.  One  c.cm.  of  a  bouillon 
culture  sterilized  by  heat  is  thought  to  be  sufficient. 
Wright  and  Semple  report  18  cases  in  which  it  was  used, 
and  by  experiment  showed  the  blood  to  be  changed  simi- 
larly to  that  of  typhoid  patients  and  convalescents.  This 
change  consisted  in  the  destruction  of  motility  and  agglu- 
tination of  the  bacilli,  as  seen  in  Widal's  reaction.  It  is 
hoped  that  we  can  gauge  the  duration  of  the  immunity 
thus  acquired  by  the  frequent  use  of  Widal's  test. 

One  of  the  most  important  and  practical  points  for  the 
physician  to  grasp  in  relation  to  the  subject  of  typhoid 
fever  is  the  highly  virulent  character  of  the  discharges 
from  the  bowels.  In  every  case  the  greatest  care  should 
be  taken  for  a  proper  disinfection  of  the  feces,  a  rigid 
attention  to  all  the  details  of  cleanliness  in  the  sick- 
room, and  the  careful  sterilization  of  all  articles  which 
are  soiled  by  the  patient.  If  country  practitioners  were 
as  careful  in  this  particular  as  they  should  be,  the  disease 
would  be  much  less  frequent  in  regions  remote  from  the 
filth  and  squalor  of  large  cities  with  their  unmanageable 
slums,  and  the  distribution  of  the  bacilli  to  villages  and 
towns,  by  watercourses  polluted  in  their  infancy,  might 
be  checked. 

1  Deutsche  med.  IVochenschrift,  1893,  No.  41. 

2  Wien.  klin.  It'ochenschrift,  July  12,  1894. 


CHAPTER  III. 
BACILLUS    COLI    COMMUNIS. 

THE  Bacillus  coli  was  first  isolated  from  human  feces 
in  1885  by  Emmerich,  who  thought  that  it  was  the  spe- 
cific cause  of  Asiatic  cholera.  Many  investigators  have 
since  studied  its  peculiarities,  until  at  the  present  time 
it  is  one  of  the  best-known  bacteria. 

It  is  habitually  present  in  the  fecal  matter  of  most  ani- 
mals except  the  horse,  and  in  water  and  soil  contaminated 


FlG.  109. — Bacillus  coli  communis,  from  an  agar-agar  culture;  x  IOOO  (Itzerott 
and  Niemann). 

with  it.  With  water  or  dust  it  gains  entrance  into  the 
mouth,  where  it  can  frequently  be  found,  and  occurs 
accidentally  in  foods  and  drinks.  During  life  the  organ- 
ism sometimes  enters  wounds  externally  from  the  surface 
of  the  body  or  internally  from  the  intestine,  and  is  a 
cause  of  suppuration — or  at  least  occurs  in  the  pus.  The 
Bacillus  pyogenes  foetidus  of  Passet  is  almost  certainly 
identical  with  it. 

389 


390  PATHOGENIC  BACTERIA. 

The  bacillus  is  rather  variable  culturally,  and  is  some- 
what polymorphic.  Probably  both  size  and  form  depend 
to  a  certain  extent  upon  the  culture-medium  on  which 
it  grows.  On  the  average,  it  measures  1-3  X  0.4-0.  y/A 
It  usually  occurs  in  the  form  of  short  rods,  but  very 
short  coccus-like  elements  and  quite  elongate  forms  are 
often  found  in  the  same  culture.  The  individual  bacilli 
are  frequently  isolated  or  in  pairs.  Chains  are  the  ex- 
ception. They  are  provided  with  flagella,  which  are 
very  variable  in  number,  generally  from  four  to  a  dozen, 
though  there  may  be  more.  It  forms  no  spores. 

The  bacillus  stains  well  with  the  ordinary  aqueous 
solutions  of  the  anilin  dyes,  but  does  not  retain  the  stain 
after  immersion  in  Gram's  solution. 

The  bacillus  is  motile,  though  in  this  particular  it  is 
subject  to  irregularity,  the  organisms  from  some  cultures 


FlG.   no. — Bacillus  coli  communis:    superficial  colony  two  days   old  upon  a 
gelatin  plate;    x  21   (Ileim). 

always  swimming  actively,  even  when  the  culture  is 
some  days  old,  others  being  exceedingly  sluggish  even 
when  voting  and  actively  growing,  and  a  few  cultures 
seem  to  consist  of  bacilli  that  do  not  move  at  all.  Fresh 
cultures  which,  when  grown  at  incubation  temperature, 
consist  of  entirely  non-motile  bacteria  are  probably  Bacil- 
lus coli  iwmohilis,  not  Bacillus  coli  communis. 

The  bacillus  is  readily  cultivated  upon  the  ordinary 


BACILLUS  CO  LI  CO  MM  UN  IS.  391 

media.  Upon  gelatin  plates  the  colonies  develop  in 
twenty-four  hours.  Those  situated  below  the  surface 
appear  round,  yellow-brown,  and  homogeneous.  As  they 
grow  older  they  increase  in  size  and  become  opaque.  ^The 
superficial  colonies  are  larger  and  spread  out  upon  the 
surface.  Their  edges  are  dentate  and  resemble  grape- 
leaves,  often  showing  radiating  ridges  suggestive  of  the 
veins  of  a  leaf.  They  may  have  a  slightly  concentric 
appearance.  The  colonies  rapidly  increase  in  size  and 
become  more  and  more  opaque.  The  gelatin  is  not 
liquefied. 

In  gelatin  punctures  the  culture,  developing  rapidly 
upon  the  surface,  and  also  in  the  needle's  track,  causes 
the  formation  of  a  nail-like  growth.  The  head  of  the 
nail  may  reach  the  walls  of  the  test-tube.  Not  infre- 
quently gas  is  formed  in  ordinary  gelatin,  and  when  i 
per  cent,  of  glucose  is  dissolved  in  the  medium  the  gas- 
production  is  often  so  copious  and  rapid  as  to  form  large 
bubbles,  which  by  their  distention  subsequently  break  it 
up  into  irregular  pieces.  Sometimes  the  gelatin  becomes 
slightly  clouded  as  the  bacilli  grow. 

Upon  agar-agar  along  the  line  of  the  inoculation  a 
grayish-white,  translucent,  smeary  growth  takes  place. 
It  is  devoid  of  any  characteristics.  The  entire  surface 
of  the  culture-medium  is  never  covered,  the  growth  re- 
maining confined  to  the  inoculation-line,  except  where 
the  moisture  of  the  condensation-fluid  allows  it  to  spread 
out  at  the  bottom.  Kruse  says  that  in  old  cultures  crys- 
tals may  form.  I  have  never  seen  them. 

Bouillon  is  soon  evenly  clouded  by  the  development 
of  the  bacteria.  Sometimes  a  delicate  pellicle  forms  upon 
the  surface.  There  is  rarely  much  sediment  in  the  cul- 
ture. 

Wiirtz  found  that  the  bacillus  produced  ammonia  in 
culture-media  free  from  sugar,  and  thus  caused  an  intense 
alkaline  reaction  in  the  culture-media.  The  cultures 
usually  give  off  an  odor  that  varies  somewhat,  but  is,  as 
a  rule,  unpleasant. 


392  PATHOGENIC  BACTERIA. 

Indol  is  formed  in  both  bouillon  and  pepton  solu- 
tions. Phenol  is  not  produced.  Litmus  added  to  the 
culture-media  is  ultimately  decolorized  by  the  bacilli. 

The  presence  of  indol  is  probably  best  determined  by 
Salkowski's  method.  To  the  culture  i  c.cm.  of  a  0.02 
per  cent,  aqueous  solution  of  potassium  nitrate  and  a 
few  drops  of  concentrated  sulphuric  acid  are  added.  If 
a  rose  color  develops,  indol  is  present. 

Nitrates  are  reduced  to  nitrites  by  the  growth  of  the 
bacillus. 

Upon  potato  the  growth  is  luxuriant.  The  bacillus 
forms  a  yellowish-brown,  glistening  layer  spreading  from 
the  line  of  inoculation  over  about  one-half  to  two-thirds 
of  the  potato.  The  color  shown  by  the  potato-cultures 
varies  considerably,  sometimes  being  very  pale,  some- 
times quite  brown.  It  cannot,  therefore,  be  taken  as  a 
characteristic  of  much  importance.  Sometimes  the  po- 
tato becomes  greenish  in  color.  Sometimes  the  growth 
on  potato  is  almost  invisible. 

In  milk  there  are  rapid  coagulation  and  acidulation, 
with  the  evolution  of  much  gas. 

The  bacillus  seems  to  require  very  little  nutriment.  It 
grows  in  Uschinsky's  asparagin  solution,  and  is  frequently 
found  living  in  river  and  well  waters. 

It  is  quite  resistant  to  antiseptics  and  germicides,  and 
grows  in  culture-media  containing  from  o.  i-o.z  per  cent. 
of  carbolic  acid.  It  lives  for  months  upon  artificial 
media. 

The  bacillus  begins  to  penetrate  the  intestinal  tissues 
almost  immediately  after  death,  and  is  the  most  frequent 
contaminating  micro-organism  met  with  in  cultures  made 
at  autopsy.  Exactly  how  it  penetrates  the  tissues  is  not 
known.  It  may  spread  by  direct  continuity  of  tissue,  or 
ria  the  blood-vessels. 

While  under  normal  conditions  a  saprophytic  bacte- 
rium, the  colon  bacillus  is  far  from  harmless.  It  not 
infrequently  is  found  in  the  pus  of  abscesses  remote  from 
the  intestine,  and  is  almost  always  found  in  suppura- 


BACILLUS  CO  LI  COM  MUNIS.  393 

tions   connected    with    the   intestines,    as,   for   example, 
appendicitis. 

It  is  a  question  whether  the  colon  bacillus  is  always 
virulent,  or  whether  it  becomes  virulent  under  abnormal 
conditions.  Klencki l  found  that  it  was  very  virulent  in 
the  ileum,  and  less  so  in  the  colon  and  jejunum,  espe- 
cially in  dogs.  He  also  found  that  the  virulence  was 
greatly  increased  in  a  strangulated  portion  of  intestine. 
Other  observers,  as  Dreyfuss,  found  that  the  colon  bacil- 
lus as  it  occurs  in  normal  feces  is  non-pathogenic.  Most 
experimenters,  however,  believe  that  pathological  con- 
ditions, such  as  disease  of  the  intestine,  ligation  of  the 
intestine,  etc.,  cause  increased  virulence. 

Adelaide  Ward  Peckham,  in  an  elaborate  study  of  the 
"  Influence  of  Environment  on  the  Colon  Bacillus,"2  con- 
cludes that  while  the  conditions  of  nutrition  and  develop- 
ment in  the  intestine  seem  to  be  most  favorable,  the  colon 
bacillus  is  ordinarily  not  virulent,  because  "  its  first  force 
is  spent  upon  the  process  of  fermentation,  and  as  long  as 
opportunities  exist  for  the  exercise  of  this  function  the 
affinities  of  this  organism  appear  to  be  strongest  in  this 
direction. 

"Moreover,  the  contents  of  the  intestine  remain  acid 
until  they  reach  the  neighborhood  of  the  colon,  and  by 
that  time  the  tryptic  peptons  have  been  formed  and 
absorbed  to  a  great  extent. 

"During  the  process  of  inflammation  in  the  digestive 
tract  a  very  different  condition  may  exist.  The  peptic  and 
tryptic  enzymes  may  be  partially  suppressed.  Fermenta- 
tion of  carbohydrates  and  proteid  foods  then  begins  in 
the  stomach,  and  continues  after  the  mass  of  food  is 
passed  on  into  the  intestine.  The  colon  bacillus  cannot, 
therefore,  spend  its  force  upon  fermentation  of  sugars, 
because  they  are  already  broken  up  and  an  alkaline  fer- 
mentation of  the  proteids  is  in  progress.  It  also  cannot 
form  peptons  from  the  original  proteids,  for  it  does  not 

1  Ann.  de  I'lnst.  Pasteur,  1895,  No.  9. 

2  Journal  of  Experimental  Medicine,  Sept.,  1897,  vol.  ii.,  No.  4,  p.  549. 


394  PATHOGENIC  BACTERIA. 

possess  this  property,  and  unless  trypsin  is  present  it  must 
be  dependent  upon  the  proteolytic  activity  of  other  bac- 
teria for  a  suitable  form  of  proteid  food.  Perhaps  these 
bacteria  form  an  albuminate  molecule,  which  like  leucin 
and  tyrosin  cannot  be  broken  up  into  indol,  and  thus 
there  might  be  caused  an  important  modification  of  the 
metabolism  of  the  colon  bacillus,  which  might  have 
either  an  immediate  or  remote  influence  upon  its  acquisi- 
tion of  disease-producing  properties,  for  our  own  experi- 
ments indicate  that  the  power  to  form  indol,  and  the 
actual  forming  of  it,  are  to  some  extent  an  indication  of 
the  possession  of  pathogenesis." 

To  the  laboratory  animals  the  colon  bacillus  is  patho- 
genic in  varying  degree.  Intraperitoneal  injections  into 
mice  cause  their  death  in  from  one  to  eight  days  if  the 
culture  is  virulent.  Guinea-pigs  and  rabbits  also  suc- 
cumb to  intraperitoneal  and  intravenous  injection.  Sub- 
cutaneous injections  are  of  less  effect,  and  in  rabbits  seem 
to  produce  abscesses  only. 

When  the  bacilli  are  injected  into  the  abdominal  cavity 
a  sero-fibrinous  or  purulent  peritonitis  occurs,  the  bacilli 
being  very  numerous  in  the  abdominal  fluids. 

The  pathogeny  of  the  colon  bacillus  is  due  to  irritating, 
chemotactic  substances  in  its  protoplasm.  The  experi- 
ments of  Pfeiffer  and  Kolle  and  Loffler  and  Abel  have 
proved  very  conclusively  that  the  poisonous  principle  is 
in,  and  cannot  by  any  means  be  separated  from  the  bodies 
of  the  bacteria. 

Frequent  transplantation  lessens  the  virulence,  passage 
through  animals  increases  it. 

Numerous  observers  have  found  that  cultures  of  the 
bacillus  obtained  from  cholera,  cholera  nostras,  and  other 
intestinal  diseases  are  much  more  pathogenic  than  those 
obtained  from  normal  feces  or  from  pus. 

Cumston,1  from  a  careful  study  of  thirteen  cases  of  sum- 
mer infantile  diarrheas,  comes  to  the  following  conclu- 
sions: 

1  International  Medical  Magazine,  Feb.,  1897. 


BACILLUS  CO  LI  COM  MUNIS.  395 

The  bacterium  coli  seems  to  be  the  pathogenic  agent 
of  the  greater  number  of  summer  infantile  diarrheas. 
This  organism  is  the  more  often  associated  with  the 

o 

streptococcus  pyogenes. 

The  virulence,  more  considerable  than  in  the  intestine 
of  a  healthy  child,  is  almost  always  in  direct  relation  to 
the  condition  of  the  child  at  the  time  the  culture  is  taken, 
and  does  not  appear  to  be  proportional  to  the  ulterior 
gravity  of  the  case. 

The  mobility  of  the  Bacterium  coli  is  in  general  pro- 
portional to  its  virulence.  The  jumping  movement, 
nevertheless,  does  not  correspond  to  an  exalted  virulence 
in  comparison  with  the  cases  in  which  the  mobility  was 
very  considerable,  without  presenting  these  jumping 
movements. 

The  virulence  of  the  Bacterium  coli  found  in  the 
blood  and  other  organs  is  identical  with  that  of  the  Bac- 
terium coli  taken  from  the  intestine  of  the  same  indi- 
vidual. 

Lesage,1  in  studying  the  enteritis  of  infants,  found  that 
in  40  out  of  50  cases  depending  upon  the  Bacillus  coli 
the  blood  of  the  patient  agglutinated  the  cultures  ob- 
tained, not  only  from  his  own  stools,  but  from  those  of 
all  the  other  cases.  From  this  uniformity  of  action  Le- 
sage very  properly  suggests  that  the  colon  bacilli  in  these 
cases  are  all  of  the  same  species. 

The  agglutinating  reaction  occurs  only  in  the  early 
stages  and  acute  forms  of  the  disease. 

It  is  not  difficult  to  immunize  an  animal  against  the 
colon  bacillus.  Loffler  and  Abel  immunized  dogs  by 
progressively  increased  subcutaneous  dosage  of  live  bac- 
teria, grown  in  solid  culture  and  distributed  through 
water.  The  injections  at  first  produced  hard  swellings. 
The  blood  of  the  immunized  animals  possessed  an  active 
bactericidal  influence  upon  the  colon  bacteria.  It  was 
not  in  the  correct  sense  antitoxic. 

In  intestinal  diseases,  such  as  typhoid,   cholera,   and 

1  Semaine  Medicale,  Oct.  20,  1897. 


396  PATHOGENIC  BACTERIA. 

dysentery,  the  bacillus  not  only  seems  to  acquire  an  un- 
usual degree  of  virulence,  but  because  of  the  existing 
denudation  of  mucous  surfaces,  etc.,  finds  it  easy  to  enter 
the  general  system,  with  the  result  of  secondary  remote 
suppurative  lesions  in  which  it  is  the  essential  factor. 
When  absorbed  from  the  intestine  it  frequently  enters 
the  kidney  and  is  excreted  with  the  urine,  causing,  inci- 
dentally, local  inflammatory  areas  in  the  kidney,  and 
occasionally  cystitis.  A  case  of  urethritis  is  reported  to 
have  been  caused  by  it. 

In  infants  cholera  infantum  may  not  infrequently  be 
caused  by  the  colon  bacillus,  though  probably  in  this 
disease  other  bacteria  play  a  very  important  role. 

The  bile-ducts  are  very  often  invaded  by  the  bacillus, 
which  may  cause  inflammation,  obstruction,  suppuration, 
or  calculous  formation. 

The  bacillus  has  also  been  met  in  puerperal  fever, 
Whickers  disease  of  the  new-born,  endocarditis,  menin- 
gitis, liver-abscess,  bronchopneumonia,  pleuritis,  chronic 
tonsillitis,  and  urethritis. 

For  the  determination  of  the  colon  bacillus  the  im- 
portant points  are  the  motility,  the  indol  reaction,  the 
milk-coagulation,  and  the  active  gas-production.  As, 
however,  all  of  these  features  are  shared  by  other  bac- 
teria to  a  greater  or  less  degree,  the  only  positive  differ- 
ential point  upon  which  very  great  reliance  can  be  placed 
is  the  immunity-reaction  of  the  serum  of  an  immunized 
animal,  which  not  only  protects  susceptible  animals  from 
the  effects  of  inoculation,  but  produces  with  fresh  cul- 
tures of  the  bacillus  exactly  the  same  reaction  as  that 
observed  in  connection  with  the  blood  and  serum  of 
typhoid  patients,  and  convalescents  and  immunized  ani- 
mals. This  reaction  has  been  considered  at  length  in 
speaking  of  typhoid  fever. 

For  the  few  who  are  convinced  that  the  colon  and 
typhoid  bacilli  are  identical,  the  fact  that  the  typhoid 
sc  ruin  is  specific  for  the  typhoid  bacillus,  and  the  colon 
serum  for  the  colon  bacillus,  with  rare  exceptions/ 


BACILLUS  CO  LI  CO  MM  UN  IS.  397 

should  be  important  evidence  of  their  separate  individ- 
uality. 

The  author  has  no  doubt  that  the  Bacillus  coli  com- 
munis  is  not  a  single  species  of  bacteria,  but  is  a  name 
applied  to  a  group  whose  individual  differences  are  thus 
far  too  similar  to  enable  us  to  differentiate  them.  This 
opinion  seems  to  be  shared  by  other  bacteriologists,  some 
of  whom  have  attempted  to  separate  the  bacillus  into 
groups,  types,  or  families. 

In  order  to  establish  a  type  species  of  the  Bacillus  coli 
communis,  Smith1  says: 

* '  I  would  suggest  that  those  forms  be  regarded  as  true 
to  this  species  which  grow  on  gelatin  in  the  form  of  deli- 
cate, bluish,  or  more  opaque,  whitish  expansions  with 
irregular  margin,  which  are  actively  motile  when  exam- 
ined in  the  hanging  drop  from  young  surface-colonies 
taken  from  gelatin  plates  which  coagulate  milk  within 
a  few  days;  grow  upon  potato,  either  as  a  rich-pale  or 
brownish-yellow  deposit,  or  merely  as  a  glistening,  barely 
recognizable  layer,  and  which  give  a  distinct  indol  reac- 
tion. Their  behavior  in  the  fermentation-tube  must 
conform  to  the  following  scheme: 

"Variety  a: 

"One  per  cent,  dextrose-bouillon  (at  37°  C.).  Total 
gas  approximately  y2\  HCO2  approximately  ji\  reaction 
strongly  acid. 

"One  per  cent,  lactose-bouillon:  as  in  dextrose-bouil- 
lon (with  slight  variations). 

"One  per  cent,  saccharose-bouillon;  gas-production 
slower  than  the  preceding,  lasting  from  seven  to  four- 
teen days.  Total  gas  about  % ;  HCO2  nearly  %.  The 
final  reaction  in  the  bulb  may  be  slightly  acid  or  alkaline, 
according  to  the  rate  of  gas-production. 

"Variety  /3: 

"  The  same  in  all  respects,  excepting  as  to  its  behavior 
in  saccharose-bouillon;  neither  gas  nor  acids  are  formed 
in  it." 

1  American  Journal  of  the  Medical  Sciences,  1895,  no,  p.  287. 


398 


PATHOGENIC  BACTERIA. 


Characteristics  for  Differentiation. 


TYPHOID  BACILLUS. 

Bacilli  usually  slender. 

Flagella  numerous  (10-20),  long,  and 
wavy. 

Growth  not  very  rapid,  not  particularly 
luxuriant. 

Upon  Eisner's  culture-medium  de- 
velops slowly,  the  colonies  remain- 
ing small. 

Upon  fresh  acid  potato  the  so-called 
"  invisible  growth  "  formerly  thought 
to  be  differential. 

Acid-production  in  whey  not  exceed- 
ing 3  per  cent.  Sometimes  slight 
in  ordinary  media,  and  sometimes 
succeeded  by  alkaline  production. 

Grows  in  media  containing  sugars 
without  producing  any  gases. 

Produces  no  indol. 

Growth  in  milk  unaccompanied  by 
coagulation. 

In  Maassen's  asparagin-glycerin  solu- 
tion the  bacillus  does  not  grow. 

Gives  the  Widal  reaction  with  the 
serum  of  typhoid  blood. 


COLON  BACILLUS. 
Bacilli  inclined  to  be  a  little  thicker. 
Flagella  fewer  (8-10). 

Growth  rapid  and  luxuriant.  This 
character  is  by  no  means  constant. 

Upon  Eisner's  medium  develops  more 
rapidly,  the  colonies  being  larger. 
(Sometimes  the  colonies  are  small 
and  remain  so.) 

Upon  potato  a  brownish-yellow,  dis- 
tinct pellicle. 

Acid-production  well  marked. 


Gas-production  well  marked. 

Indol-production  marked. 
Milk  coagulated. 

Grows  in  Maassen's  solution. 
Does  not  react  with  typhoid  blood, 


CHAPTER   IV. 
YELLOW  FEVER. 

THE  bacteriology  of  yellow  fever  has  been  studied  by 
Domingos  Freire,  Carmona  y  Valle,  Sternberg,  Havel- 
burg,  and  most  recently  by  Sanarelli. 

Sternberg,  whose  work  is  extensive  and  important, 
says:  "  Facts  relating  to  the  endemic  and  epidemic  prev- 
alence of  yellow  fever,  considered  in  connection  with 
the  present  state  of  knowledge  concerning  the  etiology 
of  other  infectious  diseases,  justify  the  belief  that  yellow 
fever  is  due  to  a  living  organism  capable  of  development 
under  favorable  local  and  meteorological  conditions  ex- 
ternal to  the  human  body,  and  of  establishing  new  cen- 
ters of  infection  when  transported  to  distant  localities." 

Sternberg,  at  the  Tenth  International  Medical  Con- 
gress (Berlin,  1890),  reported  the  study  of  42  yellow  fever 
autopsies  in  which  aerobic  and  anaerobic  cultures  were 
made  from  the  blood,  liver,  kidney,  urine,  stomach,  and 
intestines,  but  the  specific  infectious  agent  was  not  found, 
and  the  most  approved  bacteriological  methods  failed  to 
demonstrate  the  constant  presence  of  any  particular 
micro-organism  in  the  blood  and  tissues  of  yellow  fever 
cadavers.  The  micro-organism  most  frequently  encoun- 
tered was  the  Bacillus  coli  communis. 

A  few  scattered  bacilli  were  found  in  the  liver  and 
other  organs  at  the  moment  of  death,  but  when  a  portion 
of  liver  was  preserved  in  an  antiseptic  wrapper  and  kept 
for  twenty-four  to  forty-eight  hours  the  large  number  of 
bacteria  that  developed  were  of  many  varieties,  the  most 
common  being  the  Bacillus  coli  communis  and  the  Ba- 
cillus cadaveris. 

The  blood,   urine,   and  crushed  liver-tissue   obtained 


400  PATHOGENIC  BACTERIA. 

from  a  recent  autopsy  are  not  pathogenic  in  moderate 
amounts  for  rabbits  or  guinea-pigs.  Liver-tissue  pre- 
served at  28°  F.  in  an  antiseptic  wrapper  is  very  patho- 
genic for  guinea-pigs  when  injected  subcutaneously,  but 
Sternberg  found  that  this  pathogenesis  was  not  true  of 
yellow  fever  livers  only,  as  it  developed  also  in  control- 
autopsies. 

Extended  research  of  the  alimentary  canal  in  yellow 
fever  showed  the  intestine  to  contain  a  great  number  of 
bacteria,  but  no  pure  or  nearly  pure  culture  of  any  single 
species,  as  in  cholera.  Few  liquefying  bacteria  were 
found,  and  the  most  abundant  bacterium  was,  as  in 
health,  the  Bacterium  coli  communis. 

The  most  important  micro-organism  met  with  was 
Bacillus  x  (Sternberg),  which  was  isolated  by  the  culture- 
method  from  a  considerable  number  of  cases,  and  may 
have  been  present  in  all.  It  was  not  present  in  any  of 
the  control-experiments.  It  was  very  pathogenic  for  rab- 
bits when  injected  into  the  abdominal  cavity.  Sternberg 
says:  u  It  is  possible  that  this  bacillus  is  concerned  in  the 
etiology  of  yellow  fever,  but  no  satisfactory  evidence  that 
this  is  the  case  has  been  obtained  by  experiments  upon 
the  lower  animals,  and  it  has  not  been  found  in  such 
numbers  as  to  warrant  the  inference  that  it  is  the  veri- 
table infectious  agent." 

The  latest  researches  upon  yellow  fever  are  those  of 
Sanarelli.1  In  studying  the  cadavers  of  yellow  fever  San- 
arelli  found  them  either  entirely  sterile  or  universally 
invaded  by  certain  microbic  species,  such  as  the  Strepto- 
coccus pyogenes,  the  colon  bacillus,  the  protei,  etc., 
which  cannot  be  the  cause  of  the  disease.  In  the  second 
case  he  examined  he  was  fortunate  enough  to  find  what 
he  is  satisfied  is  the  specific  microbe,  the  Bacillus  ictcr- 
oides.  In  ii  autopsies  he  never  found  the  organism 
alone,  but  always  associated  with  the  ordinary  bacteria 
mentioned  above.  The  Bacillus  icteroides  must  be  sought 
for  in  the  blood  and  tissues,  and  not  in  the  gastro-intes- 

1  Brit.  MeiL  Journ.,  July  3,  1897. 


YELLOW  FEVER. 


401 


tinal  cavity.  In  the  latter  it  is  never  found.  The  isola- 
tion of  the  specific  microbe  was  only  possible  in  58  per 
cent,  of  the  cases,  and  in  some  rare  instances  may  be  ac- 
complished during  life. 

The  bacillus,  at  first  sight,  presents  nothing  morpho- 
logically characteristic.  It  is  a  small  bacillus  with 
rounded  ends,  generally  united  in  pairs  in  the  culture 
and  in  small  groups  in  the  tissues.  It'is  2-4^  in  length, 
and,  as  a  rule,  two  or  three  times  longer  than  broad  (Fig. 
in).  It  is  pleomorphous,  and  has  flagella.  By  em- 
ploying suitable  methods  it  can  be  found  in  the  organs 


FIG.   in. — Bacillus  icteroides  (Sanarelli). 

of  yellow  fever  cadavers,  usually  united  in  little  groups, 
always  situated  in  the  small  capillaries  of  the  liver,  kid- 
ney, etc.  The  best  method  of  demonstration  is  to  keep 
a  fragment  of  liver,  obtained  from  a  body  soon  after 
death,  in  the  incubator  at  37°  C.  for  twelve  hours  and 
allow  the  bacteria  to  multiply  in  the  fresh  tissue  before 
examination. 

The  bacillus  can  be  cultivated  upon  the  ordinary 
media.  Upon  gelatin  plates  it  forms  rounded,  transpar- 
ent, granular  colonies,  which  during  the  first  three  or 
four  days  present  somewhat  the  appearance  of  leukocytes. 

26 


402  PATHOGENIC  BACTERIA. 

The  granular  appearance  becomes  continuously  more 
marked,  and  usually  a  central  or  peripheric  nucleus, 
completely  opaque,  is  seen.  In  time  the  entire  colony 
becomes  opaque,  but  does  not  liquefy  gelatin. 

Stroke-cultures  on  obliquely  solidified  gelatin  exhibit 
brilliant,  opaque,  little  drops  similar  to  drops  of  milk. 

In  bouillon  it  develops  slowly,  without  either  pellicle 
or  flocculi. 

The  culture  upon  agar-agar  is  said  to  be  characteristic. 

If  grown  at  37°  C.,  the  peculiar  appearances  of  the 
colonies  do  not  develop;  but  if  the  culture  is  kept  at  20°- 
22°  C.,  the  colonies  appear  rounded,  whitish,  opaque,  and 
prominent,  like  drops  of  milk.  This  appearance  of  the 
colonies  shows  well  if  the  cultures  are  kept  for  the  first 
twelve  to  sixteen  hours  at  37°  C.,  and  afterward  at  room- 
temperature,  when  the  colonies  will  show  a  flat  central 
nucleus,  transparent  and  bluish,  surrounded  by  a  promi- 
nent and  opaque  zone,  the  whole  resembling  a  drop  of 
sealing-wax.  Sanarelli  refers  to  this  appearance  as  con- 
stituting the  diagnostic  feature  of  Bacillus  icteroides.  It 
can  be  obtained  in  twenty-four  hours. 

The  growth  upon  potato  corresponds  to  the  classic 
description  of  that  of  the  bacillus  of  typhoid  fever. 

The  bacillus  is  a  facultative  anaerobe.  It  cannot  be 
colored  by  Gram's  stain.  It  slowly  ferments  lactose, 
more  actively  ferments  glucose  and  saccharose,  but  is  not 
capable  of  coagulating  milk.  It  strongly  resists  drying, 
dies  in  water  at  60°  C.,  and  is  killed  in  seven  hours  by  the 
solar  rays.  It  can  live  for  considerable  time  in  sea-water. 

The  bacterium  is  pathogenic  for  the  majority  of  the 
domestic  animals.  All  mammals  seem  more  or  less 
sensitive  to  the  pathogenic  action  of  the  bacillus;  birds 
are  often  immune.  Guinea-pigs  are  invariably  killed  by 
either  intraperitoneal  or  subcutaneous  injection  of  o.  i 
c.t'in.  White  mice  are  killed  in  five  days;  guinea-pigs 
in  eight  to  twelve  days;  rabbits  in  four  to  five  days. 
The  morbid  changes  present  include  splenic  tumor,  hy- 
pertrophy of  the  thymus,  and  adenitis.  In  the  rabbit 


YELL  OW  FE  VER.  403 

there  are,  in  addition,  nephritis,  enteritis,  albuminuria, 
hemoglobinuria,  and  hemorrhages  into  the  body-cavities. 

The  dog  is  the  most  susceptible  animal.  When  it  is 
injected  intravenously  the  disease-process  that  results  is 
almost  immediately  manifested  with  such  violent  symp- 
toms and  such  complex  lesions  as  to  recall  the  clinical 
and  anatomical  picture  of  yellow  fever  in  the  human 
being.  The  most  prominent  symptom  in  experimental 
yellow  fever  in  the  dog  is  vomiting,  which  begins  directly 
after  the  penetration  of  the  virus  into  the  blood  and  con- 
tinues for  a  long  time.  Hemorrhages  appear  after  the 
vomiting,  the  urine  is  scanty  and  albuminous,  or  there  is 
suppression,  which  shortly  precedes  death.  Once  grave 
jaundice  was  observed. 

At  the  necropsy  the  lesions  met  are  highly  interesting, 
and  are  almost  identical  with  those  observed  in  man. 
Most  conspicuous  is  the  profound  steatosis  of  the  liver. 
The  liver-cells,  even  when  examined  fresh,  appear  com- 
pletely degenerated  into  fat,  this  appearance  correspond- 
ing to  that  found  in  fatal  cases  of  yellow  fever.  The 
same  result  may  be  obtained  by  injecting  the  liver  di- 
rectly or  through  the  abdominal  wall.  The  kidneys  are 
the  seat,  of  acute  parenchymatous  nephritis,  sometimes 
with  marked  fatty  degeneration.  The  whole  digestive 
tract  is  the  seat  of  hemorrhagic  gastro-enteritis  comparable 
in  intensity  only  to  poisoning  by  cyanid  of  potassium. 

Experiments  upon  monkeys  were  also  of  interest,  in- 
asmuch as  they  demonstrated  the  possibility  of  obtaining 
fatty  degeneration  more  extensive  than  is  observed  in 
man.  In  one  case  the  liver  was  transformed  into  a  mass 
of  fatty  substance  similar  to  wax. 

Goats  and  sheep  are  also  very  sensitive  to  the  icteroid 
virus,  and  the  lesions  described  also  occur  in  them. 

The  death  of  a  yellow  fever  victim  is  the  result  of  one 
of  three  causes: 

i.  It  may  be  due  to  the  specific  infection  principally, 
when  the  Bacillus  icteroides  is  found  in  the  cadaver  in 
a  certain  quantity  and  in  a  state  of  relative  purity. 


404  PATHOGENIC  BACTERIA. 

2.  It  may  be  due  to  the  septicemias  established  during 
the  course  of  the  disease,  the  cadaver  then  presenting  an 
almost  pure  culture  of  the  other  microbes. 

3.  It  may  be  due  in  large  measure  to  renal  insufficiency, 
when  the  cadaver  is  found  nearly  sterile. 

The  black  vomit  is  due  to  the  action  of  gastric  acidity 
upon  the  blood  which  has  extravasated  in  the  stomach  in 
consequence  of  the  toxic  products  of  the  Bacillus  icte- 
roides. 

The  Bacillus  icteroides  produces  a  toxin  the  result  of 
whose  action  corresponds  to  the  essential  symptoms  of  the 
disease.  Animals  immune  to  the  infection,  or  only  par- 
tially susceptible  to  it,  are  not  much  affected  by  the  toxin. 
Susceptible  animals,  such  as  dogs,  are  profoundly  affected. 
Ten  to  fifteen  minutes  after  injecting  the  toxin  the 
animals  experience  a  general  rigor;  abundant  lachryma- 
tion  begins,  followed  by  continued  vomiting,  first  of  food, 
then  of  mucus.  In  a  short  time  the  animals  lie  help- 
less and  extended.  Hematuria  frequently  occurs.  If  the 
dose  be  moderate,  the  dog  recovers  quickly  from  the 
violent  attack;  but  if  the  quantity  of  toxin  be  very  large 
or  repeated  on  successive  days,  it  finally  succumbs,  pre- 
senting the  anatomical  lesions  already  described  as  due  to 
infection. 

The  proofs  of  the  specificity  of  the  Bacillus  icteroides 
are  not  limited  to  the  animal  experiments  quoted.  Sana- 
relli  also  adduces  five  experimental  inoculations  upon 
men.  These  inoculations  were  not  made  with  the  bac- 
teria— i.  e.  were  not  infection  experiments — but  were 
made  with  the  filtered  sterile  toxin,  whose  action  could 
be  more  easily  controlled.  "  The  injection  of  the  filtered 
cultures  in  relatively  small  doses  reproduced  in  man 
typical  yellow  fever,  accompanied  by  all  its  imposing 
anatomical  and  symptomatological  retinue.  The  fever, 
congestions,  hemorrhages,  vomiting,  steatosis  of  the  liver, 
cephalalgia,  collapse — in  short,  all  that  complex  of  symp- 
tomatic and  anatomical  elements  which  in  their  combina- 
tion constitute  the  indivisible  basis  of  the  diagnosis  of 


YELLOW  FEVER.  405 

yellow  fever.  This  fact  is  not  only  striking  evidence  in 
favor  of  the  specific  nature  of  the  Bacillus  icteroides,  but 
it  places  the  etiological  and  pathologic  conception  of  yel- 
low fever  on  an  altogether  new  basis. ' ' 

The  discovery  of  the  Bacillus  icteroides,  and  especially 
of  its  toxin,  entirely  changes  our  view  of  the  pathology 
of  the  disease.  Instead  of  being  a  disease  of  the  gastro- 
intestinal tract,  as  one  would  conclude  from  the  symp- 
toms, "all  the  symptomatic  phenomena,  all  the  functional 
alterations,  all  the  anatomical  lesions  of  yellow  fever,  are 
only  the  consequence  of  an  eminently  steatogenous, 
emetic,  and  hemolytic  action  of  the  toxic  substances 
manufactured  by  the  Bacillus  icteroides." 

The  mode  by  which  the  Bacillus  icteroides  enters  the 
body  to  produce  the  disease  has  not  been  made  out. 
The  digestive  and  respiratory  tracts  are  the  most  likely 
routes. 

Sanarelli  points  out  that  when  it  happens  that  a  mould 
develops  near  the  Bacillus  icteroides,  the  products  of 
material  exchange  of  this  hyphomycete  or  the  transfor- 
mation effected  by  it,  are  sufficient  to  nourish  the  ba- 
cillus and  enable  it  to  live  and  multiply,  whereas  it 
would  be  otherwise  condemned  to  a  more  or  less  early 
death. 

There  seems  to  be  no  particular  mould  possessed  of  this 
power,  as  of  six  experimented  upon  all  were  capable  of 
it.  Sanarelli  is  of  the  opinion  that  in  the  holds  of  ships 
and  in  damp  places  generally  the  presence  of  moulds 
favors  the  development  of  the  Bacillus  icteroides. 

About  the  same  time  that  Sanarelli  published  his 
researches,  Havelburg  announced l  the  discovery  of  an 
entirely  different  bacillus.  Without  entering  into  a  long 
description  of  Havelburg' s  bacillus,  which  seems  to  be 
far  less  established  in  its  specificity,  the  following  are  the 
chief  characteristic  and  differential  points: 

The  bacillus  is  found  in  the  stomach  and  intestine  and 
in  the  "black  vomit."  It  is  almost  the  sole  organ- 

1  Ann.  de  r  Inst.  Pasteur,  1897. 


406  PA  THOGENIC  BA  CTERIA . 

ism  found  in  the  blood  in  the  stomach.  There  seems  to 
be  very  little  toxin  in  the  blood  of  patients  with  yellow 
fever,  30-40  c.cm.  of  blood  being  necessary  to  kill  a 
guinea-pig  when  injected  subcutaneously.  The  injection 
of  1-2  c.cm.  of  blood  from  the  stomach,  however,  caused 
death  of  the  guinea-pig.  In  its  body  an  almost  pure  cul- 
ture of  the  bacillus  of  Havelburg  was  found.  This  ex- 
periment was  repeated  twenty-one  times  without  a  failure. 

The  micro-organism  is  an  exceedingly  small  straight 
bacillus  i  p.  in  length  and  0.3-0.5  /*  in  breadth,  and  may 
be  single  or  in  pairs,  never  occurring  as  filaments.  The 
stained  specimens  are  more  deeply  colored  at  the  ends 
than  at  the  center,  so  that  the  bacillus  somewhat  resem- 
bles the  bacillus  of  fowl-cholera  and  looks  somewhat  like 
a  diplococcus.  It  has  no  flagella,  is  not  motile,  and  does 
not  seem  to  produce  spores. 

Upon  gelatin  plates  the  colonies  appear  in  twenty- 
four  hours  as  small,  round,  white  points,  and  increase 
in  size  during  the  next  twenty-four  hours.  The  older 
colonies  are  yellowish,  finely  granular  discs,  with  deli- 
cately serrated  borders.  The  gelatin  is  not  liquefied. 

In  gelatine  puncture-cultures  a  u  nail-growth  "  is  pro- 
duced, consisting  of  a  delicate  line  of  colonies  along  the 
puncture  and  a  broad  surface-growth. 

The  growth  on  agar-agar  is  not  characteristic,  as  is  that 
of  Sanarelli's  bacillus.  Bouillon  becomes  clouded  by  the 
development  of  the  organism.  Rapid  fermentation  and 
gas-production  occur  in  media  containing  sugar.  A 
grayish  growth  occurs  on  potato.  Milk  is  curdled  in 
twenty-four  hours.  The  bacillus  produces  large  quan- 
tities of  indol  and  sets  free  H2S.  Development  in  acid 
media  is  rapid.  The  organism  is  a  facultative  anaerobic. 
Guinea-pigs  and  mice  are  very  susceptible:  white  rats 
far  less  so.  Dogs  suffer  only  from  local  abscesses  at  the 
point  of  injection.  The  bacillus  rapidly  alternates  in 
virulence.  No  toxin  seems  to  be  produced  by  it. 

Havelburg  is  of  the  opinion  that  "yellow  fever  is  a 
disease  of  which  the  specific  toxic  agent  enters  the  stom- 


YELLO  W  FE  VER.  407 

ach  and  intestines,  where  it  develops.  It  is  only  excep- 
tionally and  in  small  numbers  that  it  makes  its  way  from 
these  positions  to  other  organs.  He  thinks  the  toxic  sub- 
stances formed  in  the  stomach  and  intestine  are  probably 
the  result  of  the  breaking  down  of  the  bodies  of  the  ba- 
cilli by  the  digestive  juices,  and  that  to  the  absorption  of 
these  the  various  tissue-changes  and  fatal  terminations 
are  to  be  referred. 

In  a  lengthy  and  interesting  review  and  comparison  of 
Sanarelli's  and  his  own  work,  Sternberg1  concludes  that 
the  Bacillus  icteroides  of  Sanarelli  is  identical  with  the 
Bacillus  x,  which  he  had  discovered  in  yellow  fever 
cadavers  as  early  as  1888,  and  felt  disposed  to  describe  as 
the  specific  cause  of  the  disease,  except  for  a  few  facts, 
such  as  finding  it  in  only  one-half  of  the  cases,  etc. 
Sternberg  seems  inclined  to  believe  in  Sanarelli's  work, 
and  asserts  his  intention  to  further  investigate  Bacillus  x. 
Bacillus  x  was,  however,  isolated  from  the  alimentary 
canal,  in  which  Sanarelli's  bacillus  is  said  not  to  exist, 
and  was  isolated  from  the  liver  of  a  case  of  tuberculosis, 
which  takes  away  considerable  of  the  evidence  of  its 
specificity. 

In  a  later  paper2  Sanarelli  discusses  the  validity  of 
Sternberg' s  claim  to  priority  of  discovery,  and  points  out 
a  sufficient  number  of  differences  in  the  original  descrip- 
tions of  the  organisms  to  establish  conclusively  the  in- 
dividuality of  the  Bacillus  icteroides. 

It  would  seem,  from  a  careful  consideration  of  the 
recent  literature,  that  Havelburg  had  very  little  ground 
for  considering  his  bacillus  specific,  and  that  it  is  not 
possible  for  Sternberg  to  establish  the  identity  of  the 
Bacillus  x  with  the  Bacillus  icteroides,  while  at  the  same 
time  Sanarelli's  descriptions  and  arguments  are  convinc- 
ingly in  favor  of  the  accuracy  of  his  own  work  and  the 
specificity  of  his  bacillus. 

1  Centralbl.  fur  Bakt.  und  Parasitcnk.,  Sept.  6,   1897,  Bd.  xxii.,  Nos.  6 
and  7. 

2  Ibid.,  Bd.  xxii.,  Nos.  22  and  23,  p.  668. 


408  PATHOGENIC  BACTERIA. 

Sanarelli's  labors  have  not  ceased  with  his  careful  study 
of  the  Bacillus  icteroides,  but  have  been  carried  into  the 
important  field  of  serum-therapy.  By  careful  manipula- 
tion he  has  succeeded  in  immunizing  the  horse  and  ox  to 
large  doses  of  the  bacillus,  injecting  into  a  vein  so  as  to 
prevent  the  intense  local  reaction,  and  has  found  that  the 
serum  of  these  animals  has  the  power  to  protect  guinea- 
pigs  from  lethal  doses  of  the  bacillus.  He  hopes  that 
the  serum  will  also  be  efficacious  in  the  treatment  of  yel- 
low fever  in  the  human  being. 


CHAPTER    V. 
CHICKEN- CHOLERA. 

THE  barnyards  of  Europe,  and  sometimes  of  America, 
are  occasionally  visited  by  an  epidemic  disease  which 
affects  pigeons,  turkeys,  chickens,  ducks,  and  geese,  and 
causes  almost  as  much  destruction  among  them  as  the 
occasional  epidemics  of  cholera  and  small-pox  produce 
among  men.  Rabbit-warrens  are  also  at  times  seriously 
affected  by  the  epidemic.  When  fowls  are  ill  with  the 
disease,  they  fall  into  a  condition  of  weakness  and  apathy 
which  causes  them  to  remain  quiet,  seemingly  almost 
paralyzed,  and  ruffle  up  the  feathers.  The  eyes  are 
closed  shortly  after  the  illness  begins,  and  the  birds 
gradually  fall  into  a  stupor  from  which  they  do  not 
awaken.  The  disease  leads  to  a  fatal  termination  in 
twenty-four  to  forty-eight  hours.  During  its  course 
there  is  profuse  diarrhea,  the  very  frequent  fluid,  slimy, 
grayish-white  discharges  containing  numerous  micro- 
organisms. 

The  bacilli  which  are  responsible  for  this  disease  were 
first  observed  by  Perroncito  in  1878,  and  afterward  thor- 
oughly studied  by  Pasteur.  They  are  short,  broad  bacilli 
with  rounded  ends,  sometimes  united  to  each  other, 
with  the  production  of  moderately  long  chains  (Fig.  112). 
Pasteur  at  first  regarded  them  as  cocci,  because  when 
stained  with  a  penetrating  anilin  dye  the  poles  stain 
intensely,  but  a  narrow  space  between  them  remains 
almost  uncolored.  This  peculiarity  is  very  marked,  and 
sharp  observation  is  required  to  observe  the  outline  of 
the  intermediate  substance.  The  bacillus  does  not  form 
spores,  and  does  not  stain  by  Gram's  method.  When 

409 


4io 


PATHOGENIC  BACTERIA. 


examined  in  the  living  condition  it  is  found  to  be  non- 
motile.1 

The  bacillus  readily  succumbs  to  the  action  of  heat 
and  dryness.  The  cultures  upon  gelatin  plates  after 
about  two  days  appear  as  irregular,  small,  white  points. 
The  deep  colonies  reach  the  surface  slowly,  and  do  not 
attain  any  considerable  size.  The  gelatin  is  not  lique- 
fied. The  microscope  shows  the  colonies  to  be  irregularly 


FlG.    112. — Bacillus  of  chicken-cholera,  from  the  heart's  blood  of  a  pigeon; 
x  1000  (Frankel  and  Pfeiffer). 

rounded  disks  with  distinct  smooth  borders.  The  color 
is  yellowish-brown,  and  the  contents  are  granular.  Some- 
times there  is  a  distinct  concentric  arrangement. 

In  gelatin  puncture-cultures  a  delicate  white  line  occurs 
along  the  entire  path  of  the  wire.  When  viewed  through 
a  lens  this  line  is  seen  to  consist  of  aggregated  mi- 
nute colonies.  Upon  the  surface  the  development  is 

1  Most  authorities  state  that  the  bacillus  is  not  motile,  but  Thoinot  and  Mas- 
uelin  assert  that  it  is  so.  Precis  tic  Microi>ie,  2d  ed.,  1893. 


CHICKEN  CHOLERA .  411 

much  more  marked,  so  that  the  growth  resembles  a  nail 
with  a  pretty  good-sized  flat  head.  If,  instead  of  a  punc- 
ture, the  inoculation  be  made  upon  the  surface  of  ob- 
liquely solidified  gelatin,  a  much  more  pronounced  growth 
takes  place,  and  along  the  line  of  inoculation  a  dry, 
granular  coating  is  formed.  This  growth  is  quite  similar 
to  that  upon  agar-agar  and  blood-serum,  which  growths 
are  white,  shining,  rather  luxuriant,  and  devoid  of  char- 
acteristics. No  growth  occurs  in  the  absence  of  oxygen. 

Upon  potato  no  growth  occurs  except  at  the  incubation 
temperature.  It  is  a  very  insignificant,  yellowish-gray, 
translucent  film. 

The  introduction  of  cultures  of  this  bacillus  into  the 
tissues  of  chickens,  geese,  pigeons,  sparrows,  mice,  and 
rabbits  is  sufficient  to  produce  fatal  septicemia.  Feeding 
chickens,  pigeons,  and  rabbits  with  material  infected 
with  the  bacillus  is  also  sufficient  to  produce  the  disease 
with  pronounced  intestinal  lesions.  Guinea-pigs  usually 
seem  immune,  though  they  succumb  to  very  large  doses, 
especially  when  given  intraperitoneally. 

The  autopsy  shows  that  when  the  bacilli  are  intro- 
duced subcutaneously  a  true  septicemia  results,  with  the 
addition  of  a  hemorrhagic  exudate  and  gelatinous  infil- 
tration at  the  seat  of  inoculation.  The  liver  and  spleen 
are  enlarged;  circumscribed,  hemorrhagic,  and  infiltrated 
areas  occur  in  the  lungs  ;  the  intestine  shows  an  intense 
inflammation  with  red  and  swollen  mucosa,  and  oc- 
casional ulcers  following  small  hemorrhagic  spots.  Peri- 
carditis is  of  frequent  occurrence.  The  bacilli  are  found 
in  all  the  organs.  If,  on  the  other  hand,  the  disease  has 
been  produced  by  feeding,  the  bacilli  are  chiefly  to  be 
found  in  the  intestine.  Pasteur  found  that  when  pigeons 
were  inoculated  into  the  pectoral  muscles,  if  death  did 
not  come  on  rapidly,  portions  of  the  muscle  (sequestra) 
underwent  degeneration  and  appeared  anemic,  indurated, 
and  of  a  yellowish  color. 

The  bacillus  of  chicken-cholera  is  one  whose  peculiar- 
ities can  be  made  use  of  for  protective  vaccination. 


412  PA  THOGENIC  BA  CTERIA. 

Pasteur  discovered  that  when  cultures  are  allowed  to 
remain  undisturbed  for  several  months,  their  virulence 
is  greatly  lessened,  and  new  cultures  planted  from  these 
are  also  attenuated.  When  chickens  are  inoculated  with 
such  cultures,  no  other  change  occurs  than  a  local  in- 
flammatory reaction  by  which  the  birds  are  protected 
against  virulent  bacilli.  From  this  observation  Pasteur 
worked  out  a  system  of  protective  vaccination  in  which 
fowls  can  first  be  inoculated  with  very  weak,  then  with 
stronger,  and  finally  with  highly  virulent  cultures,  with 
a  resulting  protection  and  immunity.  Unfortunately, 
the  method  is  too  complicated  to  be  very  practical.  Use 
has,  however,  been  made  of  the  ability  of  this  bacillus 
to  kill  rabbits,  and  in  Australia,  where  they  are  pests, 
they  are  being  exterminated  by  the  use  of  bouillon  cul- 
ture. It  is  estimated  that  two  gallons  of  bouillon  culture 
will  destroy  20,000  rabbits  irrespective  of  infection  by 
contagion. 

The  bacillus  of  chicken-cholera  seems  not  only  to  be 
specific  for  that  disease,  but  seems  able,  when  properly 
introduced  into  various  other  animals,  to  produce  several 
different  diseases.  Indeed,  no  little  confusion  has  arisen 
in  bacteriology  by  the  description  of  what  is  now  pretty 
generally  accepted  to  be  this  very  bacillus  under  the 
various  names  of  bacillus  of  rabbit-septicemia  (Koch), 
Bacillus  cuniculicida  (Fliigge),  bacillus  of  swine-plague 
(Loffler  and  Schiitz),  bacillus  of  lt  Wildseuche"  (Hiippe), 
bacillus  of  "  Biiffelseuche  "  (Oriste-Armanni),  etc. 


CHAPTER   VI. 
HOG-CHOLERA. 

THE  bacillus  of  hog-cholera  (Bacillus  suipestifer)  was 
first  found  by  Salmon  and  Smith,1  but  was  for  a  long  time 
confused  with  the  bacillus  of  "swine-plague,"  which  it 
closely  resembles  and  with  which  it  frequently  occurs. 
It  is  a  member  of  the  group  of  which  the  Bacillus  coli 
communis  may  be  taken  as  a  type.  Since  the  careful 
studies  of  Smith,1  however,  the  claims  of  the  discoverers 
that  the  bacillus  of  hog-cholera  is  a  separate  and  specific 
organism  can  hardly  be  doubted. 

Hog-cholera,  or  u  pig  typhoid,"  as  the  English  call  it, 
is  a  common  epidemic  disease  of  swine,  which  at  times 
kills  90  per  cent,  of  the  infected  animals,  and  thus  causes 
immense  loss,  to  breeders.  Salmon  estimates  that  the 
annual  losses  from  this  disease  in  the  United  States 
range  from  $10,000,000  to  $25,000,000. 

The  disease  is  particularly  fatal  to  young  pigs.  The 
symptoms  are  not  very  characteristic,  and  the  animals 
often  die  suddenly  without  having  appeared  particularly 
ill,  or  after  seeming  ill  but  a  few  hours.  The  symptoms 
consist  of  fever  (io6°-io7°  F.),  unwillingness  to  move, 
and  more  or  less  loss  of  appetite.  The  animals  may  ap- 
pear stupid  and  dull,  and  have  a  tendency  to  hide  in  the 
bedding  and  remain  covered  by  it.  The  bowels  may  be 
normal  or  constipated  at  the  beginning  of  the  attack, 
but  later  there  is  generally  a  liquid  and  fetid  diarrhea, 
abundant,  exhausting,  and  persisting  to  the  end.  The 
eyes  are  congested  and  watery,'  the  secretion  drying  and 

1  Reports  of  the  Bureau  of  Animal  Industry ^  1885—91. 

z  Centralbl.  fur  Bakt.  und  Parasitenk.,  Bd.  ix.,  Nos.  8,  9,  and  10,  March 
2,  1897. 

413 


PATHOGENIC  BACTERIA. 

gluing  the  lids  together.  The  breathing  is  rapid,  and 
there  may  be  cough.  Occasionally  there  is  an  eruption 
with  crusts  or  scabs  of  various  sizes  on  the  skin,  which 
is  often  congested.  The  animal  becomes  weak,  stands 
with  arched  back  and  drawn  abdomen,  and  walks  with  a 
weak,  tottering  gait. 

The  course  of  this  disease  varies  from  one  or  two  days 
to  two  or  three  weeks. 

At  post-mortem  examination  petechiae,  ecchymoses,  and 
extravasations  of  blood  into  the  tissues  are  found  to  be  com- 
mon and  form  one  of  the  principal  changes  in  the  acute 


FIG.  113.— Bacillus  of  hog-cholera,  showing  flagella. 

form  of  the  disease.  The  spleen  is  enlarged  to  two  or  four 
times  its  normal  size,  and  is  soft  and  engorged  with  blood. 
The  extravasations  of  blood  are  common  in  the  lym- 
phatic glands,  beneath  the  serous  membranes  of  the 
thorax  and  abdomen,  and  particularly  along  the  intes- 
tines; on  the  surface  of  the  lungs  and  kidneys  and  in 
their  substance.  The  contents  of  the  intestine  are  some- 
times covered  with  clotted  blood.  In  the  subacute  form 
of  the  disease  the  principal  changes  are  found  in  the 
large  intestine,  and  consist  of  ulcers  which  appear  as 
circular,  slightly  projecting  masses  varying  in  color  from 


HOG-CHOLERA.  415 

yellowish  to  black.  Occasionally  these  ulcers  are  slightly 
depressed  in  outline.  When  cut  across  they  are  found  to 
consist  of  a  firm,  solid  growth  extending  nearly  through 
the  intestinal  wall.  They  are  most  frequent  in  the 
cecum,  upper  half  of  the  colon,  and  on  the  ileocecal 
valve.  In  the  chronic  form  of  the  disease  the  spleen  is 
rarely  enlarged. 

u  In  hog-cholera  the  first  effect  of  the  disease  is 
believed  to  be  upon  the  intestines,  with  secondary  inva- 
sion of  the  lungs." 

The  most  characteristic  lesions  of  the  disease  are  the 
petechiae  and  ecchymoses,  the  ulcerations  of  the  large 
intestine  (Fig.  113),  and  the  collapse  and  occasional  bron- 
chopneumonic  changes  in  the  lung. 

The  kidneys  are  nearly  always  affected,  the  urine  con- 
taining albumin  and  tube-casts. 

The  specific  bacillus  of  hog-cholera  was  secured  by 
Smith  from  the  spleens  of  more  than  500  hogs.  It 
occurs  in  all  the  organs  and  has  also  been  cultivated 
from  the  urine. 

The  organisms  appear  as  short  rods  with  rounded 
ends,  1.2-1.5  p.  long  and  0.6-0.7  P  ^n  breadth.  They  are 
very  actively  motile.  No  spore-production  has  ever  been 
observed.  In  general  the  bacillus  resembles  in  appearance 
that  of  typhoid  fever.  It  stains  readily  by  the  ordinary 
methods,  but  not  by  Gram's  method. 

The  bacilli  possess  numerous  long  flagella,  easily 
demonstrable  by  the  usual  methods  of  staining  (Fig. 
114). 

No  trouble  is  experienced  in  cultivating  the  bacilli, 
which  grow  well  in  all  the  media. 

Upon  gelatin  plates  the  colonies  become  visible  in 
twenty-four  to  forty-eight  hours;  the  deeper  ones  spher- 
ical with  sharply  defined  borders.  The  surface  is  brown- 
ish by  reflected  light,  and  is  without  markings.  They 
are  rarely  larger  than  0.5  mm.  in  diameter  and  are  homo- 
geneous throughout.  The  superficial  colonies  have  little 
tendency  to  spread  upon  the  gelatin.  Their  borders  may 


4 1 6  PA  THOGENIC  BA  CTERIA. 

be  circular  and  rounded,  or  irregular.  They  are  said 
rarely  to  reach  a  greater  diameter  than  2  mm.  The  gela- 
tin is  not  liquefied.  There  is  nothing  distinctly  charac- 
teristic about  the  appearance  of  the  colonies. 

Upon  agar-agar  the  superficial  colonies  attain  a  diam- 


FlG.  114. — Ulceration  of  the  intestine  in  a  typical  case  of  swine-fever 
(Crookshank). 

eter  of  4  mm.  and  have  a  gray  translucent  appearance  with 
polished  surface.     They  are  round  and  slightly  arched. 

In  gelatin  punctures  the  growth  takes  the  form  of  a 
nail  with  a  flat  head.  There  is  nothing  characteristic 
about  it.  The  growth  in  the  puncture  shows  it  to  be  an 
optional  anaerobe. 


HOG-CHOLERA.  417 

Linear  cultures  upon  agar-agar  present  a  translucent, 
rather  circumscribed,  grayish,  smeary  layer. 

Upon  potato  a  yellowish  coating  is  formed,  especially 
when  the  culture  is  kept  in  the  thermostat. 

Bouillon  made  with  or  without  pepton  is  clouded  in 
twenty-four  hours.  When  the  culture  is  allowed  to  stand 
for  a  couple  of  weeks  without  being  disturbed  a  thin 
surface-growth  can  be  observed. 

Milk  is  an  excellent  culture-medium,  but  is  not  visibly 
changed  by  the  growth  of  these  bacteria.  Its  reaction 
remains  alkaline. 

The  hog-cholera  bacillus  is  a  copious  gas-producer, 
capable  of  breaking  up  sugars  into  CO2,  H,  and  an  acid, 
which,  formed  late,  eventually  checks  its  further  devel- 
opment. No  indol  and  no  phenol  are  formed  in  the 
culture-media. 

The  bacillus  is  hardy.  Smith  found  it  vital  after  being 
kept  dry  for  four  months.  It  ordinarily  dies  sooner,  how- 
ever. The  thermal  death-point  is  54°  C.,  maintained  for 
sixty  minutes. 

The  bacillus  is  markedly  pathogenic  for  animals. 
Small  quantities  introduced  subcutaneously  into  rabbits 
or  mice  kill  them  in  from  seven  to  twelve  days.  The 
animal  appears  quite  well  for  three  or  four  days,  then 
begins  to  sit  quietly  in  the  cage  and  eat  but  little,  or 
refuses  to  eat  at  all,  until  death  takes  place. 

In  Smith's  experiments  one-four-millionth  of  a  cubic 
centimeter  of  a  bouillon  culture  injected  subcutaneously 
into  a  rabbit  was  sufficient  to  cause  its  death.  Before 
death  the  temperature  abruptly  rises  2°-3°  C.,  and  re- 
mains high  until  death.  Larger  quantities  may  kill  in 
five  days.  Injected  intravenously  in  small  doses  the  ba- 
cillus may  cause  death  in  forty-eight  hours. 

When  the  animal  is  subjected  to  a  postmortem  exam- 
ination the  spleen  is  found  enlarged,  firm,  and  dark  red 
in  color.  The  liver  is  found  to  contain  small  yellowish- 
white  necrotic  areas  which  sometimes  occur  in  one,  some- 
times in  several  acini,  and  not  infrequently  surround  the 

27 


418  PATHOGENIC  BACTERIA. 

interlobular  veins.  The  kidneys  are  acutely  inflamed 
and  the  urine  is  albuminous.  The  heart-rnuscle  is 
spotted,  gray,  and  fatty.  In  the  intestinal  tract  the  pic- 
ture of  the  disease  will  be  found  to  vary  according  to  its 
duration. 

The  contents  of  the  small  intestine  are  yellowish, 
watery,  and  mucous;  Peyer's  glands  are  enlarged.  In 
the  neighborhood  of  the  pylorus,  ecchymoses  and  exten- 
sive extravasations  of  blood  are  common.  The  bacilli 
are  found  in  all  of  the  organs. 

The  house  mouse  is  very  susceptible  to  the  disease; 
guinea-pigs  much  less  so,  -fa  c.cm.  of  a  virulent  cul- 
ture often  being  required  to  kill  them.  Pigeons  are  still 
more  refractory,  and  Smith  found  that  ^  c.cm.  of  a 
bouillon  qulture  injected  into  the  breast-muscles  was 
required  to  kill  them. 

In  spite  of  the  fact  that  hog-cholera  is  a  disease  of 
swine,  and  that  it  is  from  dead  swine  that  the  bacilli  are 
obtained,  these  animals  are  not  very  easily  affected  arti- 
ficially. They  snow  no  symptoms  when  injected  subcti- 
taneously,  but  almost  invariably  die  after  intravenous 
injection  of  1-2  c.cm.  of  a  virulent  culture. 

Smith  found  that  feeding  with  200-300  c.cm.  of  a 
bouillon  culture  after  a  day's  fasting,  or  with  small  quan- 
tities administered  daily,  would  also  cause  death,  with  a 
widespread  diphtheritic  inflammation  of  the  stomach  and 
colon.  Feeding  with  the  organs  of  dead  hogs  produces 
the  same  lesions  as  the  administration  of  the  culture. 

As  early  as  1886  Salmon  and  Smith  found  it  possible 
to  produce,  in  both  very  and  partly  susceptible  animals, 
immunity  to  hog-cholera  by  gradually  accustoming  them 
to  increasing  doses  of  the  bacteria.  DeSchweinitz  iso- 
lated from  cultures  of  the  bacteria  two  toxic  substances, 
a  ptomain  (sucholo-toxin)  and  an  albumose  (sucholo-albu- 
min),  together  with  cadaverin  and  methylamin.  With 
these  substances  he  seems  to  have  been  able  to  produce 
immunity.  Selander  and  Metschnikoff  found  that  im- 
munity could  be  produced  more  quickly  by  the  use  of 


HOG-CHOLERA.  419 

blood  of  infected  rabbits  exposed  to  58°  C.  This  blood 
was  found  to  be  exceedingly  toxic. 

DeSchweinitz l  found  that  the  introduction  of  progress- 
ingly  increased  amounts  of  cultures  into  cows  caused  the 
development  in  them  of  an  antitoxic  substance  capable 
of  protecting  guinea-pigs  from  the  disease. 

Working  in  my  laboratory,  Pitfield2  has  found  .that 
after  a  single  injection  of  a  sterilized  bouillon  culture  of 
the  bacillus  into  the  horse,  the  serum,  which  has  origin- 
ally slight  agglutinative  reactive  power,  is  so  changed  as 
to  show  a  decided  reaction.  If  the  horse  be  immunized 
to  large  doses  of  such  sterile  cultures,  the  serum  reaction 
becomes  so  marked  that  with  a  dilution  of  i  :  10,000  a 
typical  reaction  occurs  in  sixty  minutes. 

According  to  this  experiment,  in  doubtful  cases  the 
use  of  this  reaction  should  greatly  facilitate  the  differen- 
tiation of  the  bacillus  of  hog-cholera  from  similar  ba- 
cilli. 

1  Cenlralbl.  f.  Bakt.  u.  Parasitenk.,  xx.,  p.  573- 

2  Microscopical  Bulletin,  1897,  p.  35. 


CHAPTER  VII. 
SWINE-PLAGUE. 

THE  bacillus  of  swine-plague,  or  the  Bacillus  suisepti- 
cus  of  Loffler  and  Schutz,  and  Salmon  and  Smith,  so 
closely  resembles  that  of  chicken-cholera  that  it  is  easily 
confounded  with  it,  and,  indeed,  at  one  time,  they  were 
thought  to  be  identical.  The  species  has,  however,  suf- 
ficient well-marked  characters  to  make  its  differentiation 
clear  (Fig.  115). 

Swine-plague   is  a  rather  common   and   exceedingly 


FIG.  115. — Bacillus  of  swine-plague  (from  photograph  by  E.  A.  de  Schweinitz). 

fatal  disease.  It  occurs  alone  or  in  combination  with 
hog-cholera  (q.  v.\  and  because  of  the  lack  of  suffi- 
ciently well-characterized  symptoms — sick  hogs  appear- 
ing more  or  less  alike— it  is  often  mistaken  for  that 
disease.  The  confusion  resulting  from  the  mixed  cases 
makes  it  impossible  to  determine  exactly  how  fatal  swine- 
plague  may  be  in  uncomplicated  cases. 

420 


SWINE-PLAGUE.  421 

The  symptoms  of  swine-plague,  while  closely  resem- 
bling those  of  hog-cholera,  may  differ  from  them  in  the 
existence  of  cough,  swine-plague  being  prone  to  affect  the 
lungs  and  oppress  the  breathing,  which  becomes  frequent, 
labored,  and  painful,  and  associated  with  frequent  cough, 
while  hog-cholera  chiefly  presents  intestinal  symptoms. 

The  course  of  the  disease  is  usually  rapid,  a  fatal  result 
often  occurring  in  one  or  two  days. 

At  autopsy  the  lungs  are  often  found  inflamed,  and 
contain  numerous  small,  pale,  necrotic  areas,  and  some- 
times large  cheesy  masses  one  or  two  inches  in  diameter. 
Inflammations  of  the  serous  membranes  affecting  the 
pleura,  pericardium,  and  peritoneum,  and  associated  with 
fibrinous  inflammatory  deposits  on  the  surfaces,  are  com- 
mon. There  may  be  congestion  of  the  mucous  mem- 
brane of  the  intestines,  particularly  of  the  large  intestine, 
or  the  disease  in  this  region  may  be  an  intense  croupous 
inflammation  with  the  formation  of  a  fibrinous  exudative 
deposit  on  the  surface. 

A  hemorrhagic  form  of  the  disease  is  said  to  be  com- 
mon in  Europe,  but,  according  to  Salmon,  is  rare  in  the 
United  States. 

The  bacillus  of  swine-plague  much  resembles  that  of 
hog-cholera,  and  not  a  little  that  of  chicken-cholera.  It 
is  a  short  organism,  rather  more  slender  than  its  con- 
geners, not  possessed  of  flagella,  and  is  incapable  of  move- 
ment and  produces  no  spores.  Its  vitality  is  low,  and 
it  is  easily  destroyed.  Salmon  says  that  it  soon  dies  in 
water  or  by  drying,  and  that  the  temperature  for  its 
growth  must  be  more  constant  and  every  condition  of 
life  more  favorable  than  for  the  hog-cholera  germ.  This 
germ  is  said  to  be  widely  distributed  in  nature,  and  is 
probably  present  in  every  herd  of  swine,  though  not 
pathogenic  except  when  its  virulence  has  been  increased 
or  the  resistance  of  the  animals  diminished  by  some  un- 
usual conditions. 

In  its  growth  the  bacillus  of  swine-plague  is  an  optional 
anaerobic  organism. 


422  PATHOGENIC  BACTERIA. 

In  general,  its  appearance  in  culture-media  is  very 
similar  to  that  of  the  bacillus  of  hog-cholera.  Kruse, 
however,1  points  out  that  when  the  bacillus  grows  in 
bouillon  the  liquid  remains  clear  on  account  of  the  for- 
mation of  a  flocculent,  stringy  sediment.  Upon  ordi- 
nary acid  potato  the  bacillus  does  not  grow,  but  if  the 
reaction  of  the  medium  be  alkaline  a  grayish-yellow  patch 
is  formed.  In  its  growth  in  milk  slight  acidity  is  pro- 
duced, but  the  milk  is  not  coagulated  and  the  litmus 
color  added  to  it  is  not  decolorized. 

The  bacillus  stains  by  the  ordinary  methods,  some- 
times only  at  the  poles,  then  resembling  very  closely  the 
bacillus  of  chicken-cholera.  It  is  not  colored  by  Gram's 
method. 

The  pathogenesis,  while  similar  to  that  of  the  hog- 
cholera  bacillus,  presents  some  marked  differences,  espe- 
cially in  regard  to  the  seat  of  the  local  manifestations, 
to  which  attention  has  already  been  called,  and  in  the 
duration  of  the  disease,  which  is  much  shorter.  There 
is  also  considerable  resemblance  to  the  bacillus  of  chicken- 
cholera  in  pathogenesis,  but  the  local  reaction  following 
injection  of  the  culture  partakes  of  the  nature  of  a  hemor- 
rhagic  edema,  which  is  not  present  in  chicken-cholera,  and 
the  cases  often  exhibit  fatty  metamorphosis  of  the  liver. 

Rabbits,  mice,  and  small  birds  are  all  very  susceptible 
to  the  disease,  generally  dying  of  septicemia  in  twenty- 
four  hours;  guinea-pigs  are  less  susceptible,  except  the 
very  young  animals,  which  die  without  exception.  Chick- 
ens are  more  immune,  but  usually  succumb  to  large  doses. 
Hogs  die  after  subcutaneous  injection  of  the  bacilli,  and 
suffer  from  marked  edema  at  the  point  of  injection,  and 
septicemia.  If  injected  into  the  lung,  a  pleuropneumonia 
with  multiple  necrotic  areas  in  the  lung  follows.  In 
these  cases  the  spleen  is  not  much  swollen,  there  is  slight 
gastro-intestinal  catarrh,  and  the  bacilli  are  present  every- 
where in  the  blood. 

Animals  cannot  be  infected  by  feeding. 

1  FlUgge's  Mikroorganismen,  p.  419,  1896. 


CHAPTER    VIII. 

TYPHUS   MURIUM. 

THE  Bacillus  typhi  murium  (Fig.  116),  which  created 
havoc  among  the  mice  in  his  laboratory,  causing  most 
of  them  to  die,  was  discovered  by  Loffler  in  1889.  It 
is  a  short  organism,  somewhat  resembling  the  bacillus 
of  chicken-cholera.  It  is  rather  variable  in  its  dimen- 
sions, and  often  grows  into  long,  flexible  filaments.  No 


FlG.    Il6. — Bacillus   typhi   murium,  from   agar-agar;     x    1000   (Itzerott   and 

Niemann). 

sporulation  has  been  observed.  It  is  a  motile  organism, 
with  numerous  flagella,  like  those  of  the  typhoid-fever 
bacillus.  It  stains  well  with  the  ordinary  dyes,  but 
rather  better  with  Loffler's  alkaline  methylene  blue. 

Upon  gelatin  plates  the  deep  colonies  are  at  first  round, 
slightly  granular,  transparent,  and  grayish.  Later  they 
become  yellowish-brown  and  granular.  Superficial  col- 
onies are  similar  to  those  of  the  typhoid  bacillus.  In 

423 


424  PATHOGENIC  BACTERIA. 

gelatin  punctures  there  is  no  liquefaction.  The  growth 
takes  place  upon  the  surface  principally,  where  a  grayish- 
white  mass  slowly  forms. 

Upon  agar-agar  a  grayish-white  development  devoid 
of  peculiarities  occurs. 

Upon  potato  a  rather  thin  whitish  growth  may  be 
observed  after  a  few  days. 

The  bacillus  grows  well  in  milk,  with  the  production 
of  an  acid  reaction,  but  without  coagulation. 

The  organism  is  pathogenic  for  mice  of  all  kinds, 
which  succumb  in  from  one  to  two  days  when  inoculated 
subcutaneously,  and  in  eight  to  ten  or  twelve  days  when 
fed  upon  material  containing  the  bacillus.  The  bacilli 
multiply  rapidly  in  the  blood-  and  lymph-channels,  and 
cause  death  from  a  general  septicemia. 

Loffler  expressed  the  opinion  that  this  bacillus  might 
be  of  use  in  ridding  infested  premises  of  mice,  and  the 
results  of  its  use  for  this  purpose  have  been  highly  satis- 
factory. He  has  succeeded  in  ridding  a  field  so  infested 
as  to  be  useless  for  agricultural  purposes  by  saturating 
some  bread  with  bouillon  cultures  of  the  bacillus  and 
distributing  it  near  the  holes  inhabited  by  the  mice. 
The  bacilli  that  were  eaten  by  the  mice  not  only  killed 
them,  but  also  infected  others  which  ate  the  dead  bodies 
of  the  first  victims,  and  so  the  extermination  progressed 
until  scarcely  a  mouse  remained  in  the  field.  In  discuss- 
ing the  practical  applicability  of  the  employment  of  cul- 
tures of  this  bacillus  for  the  destruction  of  field-mice, 
Brunner1  calls  attention  to  certain  conditions  that  are 
requisite  for  a  satisfactory  result,  (i)  It  is  necessary, 
first  of  all,  to  attack  rather  extensive  areas  of  the  invaded 
territory,  and  not  to  attempt  to  destroy  the  mice  of  a 
small  field  into  which  an  indefinite  number  of  fresh 
animals  may  immediately  come  from  the  surrounding 
fields.  The  country-people,  who  are  the  sufferers,  should 
combine  their  efforts  so  as  to  extend  the  benefits  widely. 
(2)  The  preparation  of  the  cultures  is  a  matter  of  im- 

1  Centralbl.f.  Bakt.  u  rarasiu-iit.,  Jan.  19,  1898,  Bd.  xxiii.,  No.  2. 


TYPHUS  MURIUM.  425 

portance.  Agar-agar  cultures  are  best,  as  being  most 
readily  transportable.  They  are  broken  up  in  water  and 
well  stirred,  and  the  liquid  poured  upon  a  large  num- 
ber of  small  pieces  of  broken  bread.  These  are  next  to 
be  distributed  with  reasonable  care.  Instead  of  being 
carelessly  scattered  over  the  ground,  they  should  be 
dropped  into  the  fresh  mouse-holes,  and  pushed  suffici- 
ently far  in  to  escape  the  effects  of  sunlight  upon  the 
bacilli.  Attention  should  be  paid  to  holes  in  walls, 
under  railway  tracks,  etc.  and  other  places  where  mice 
live  in  greater  freedom  from  disturbance  than  in  the 
fields.  (3)  The  attempted  eradication  of  the  mice  should 
be  begun  at  a  time  of  year  when  the  natural  food  is  not 
plenty.  By  observing  these  precautions  the  mice  can  be 
eradicated  with  certainty,  usually  in  a  period  of  time  not 
exceeding  eight  to  twelve  days.  For  this  purpose,  in  the 
course  of  two  years,  no  less  than  250,000  cultures  were 
distributed  from  the  Bacteriological  Laboratory  of  the 
Tierarznei  Institut  in  Vienna.  The  bacilli  are  not 
pathogenic  for  the  animals,  such  as  the  fox,  weasel, 
ferret,  etc.  that  feed  upon  the  mice,  do  not  affect  man 
in  any  way,  and  so  seem  to  occupy  a  useful  place  in 
agriculture  by  destroying  the  little  but  almost  invincible 
enemies  of  the  grain. 


CHAPTER   IX. 
MOUSE-SEPTICEMIA. 

IN  1878,  during  his  investigations  upon  the  infectious 
traumatic  diseases,  Koch  observed  that  when  a  minute 
amount  of  putrid  blood  or  of  meat-infusion  was  injected 
into  mice  the  animals  died  of  a  septicemia  caused  by  the 
multiplication  in  their  blood  of  a  minute  bacillus  to 
which  he  gave  the  name  u  Bacillus  der  Mausesepticamie  " 
(Fig.  117). 


FIG.  117. — Bacillus  of  mouse-septicemia,  from  the  blood  of  a  mouse;  x  1000 
(Frankel  and  Pfeiffer). 

In   1885  *ne  bacillus  was  again  brought  into  promi- 
nence by  Loffler  and  Schiitz,  who  found  a  very  similar, 


MOUSE-SEPTICEMIA.  427 

perhaps  identical,  organism  in  the  erysipelatous  disease 
which  attacks  the  swine  of  many  parts  of  Europe. 

There  seem  to  be  certain  slight  morphological  and 
developmental  differences  between  these  two  organisms, 
but  Baumgarten,  Gunther,  Sternberg,  and  others  have 
regarded  them  as  insufficient  for  the  formation  of  sepa- 
rate species,  and  have  boldly  described  the  organisms  as 
identical,  while  Lorenz  has  shown  that  inimunity  pro- 
duced in  the  rabbit  by  one  bacillus  protects  against  the 
other.  The  described  differences  are,  indeed,  so  very 
small  that  I  think  it  well  to  follow  in  the  path  of  the  ob- 
servers mentioned,  pointing  out  in  the  description  such 
points  of  difference  as  may  arise. 

The  bacilli  are  extremely  minute,  measuring  about 
i.o  x  0.2  p.  (Sternberg).  Fliigge,  Frankel,  and  Eisenberg 
find  the  Bacillus  erysipelas  suis  somewhat  shorter  and 
stouter  than  that  of  mouse-septicemia :  there  seems  to 
be  a  division  of  opinion  upon  this  point. 

Sporulation  has  been  described  by  some  observers,  but 
nothing  definite  seems  to  be  known  upon  this  point. 

Motility  is  ascribed  by  some  (Schottelius  and  Frankel) 
to  the  Bacillus  erysipelas  suis,  and  is  denied  to  the  bacillus 
of  mouse-septicemia  by  others.  The  truth  seems  to  be 
that  the  motility  of  both  organisms  is  a  matter  of  doubt. 

No  flagella  have  been  demonstrated  upon  the  bacillus. 
It  grows  quite  well  both  at  the  room-temperature  and  at 
the  temperature  of  incubation.  It  can  grow  well  with  or 
without  oxygen,  but  perhaps  flourishes 
a  little  better  without  than  with  it.  It 
is  killed  by  a  temperature  of  52°  C.  in 
fifteen  minutes. 

The  colonies  upon  gelatin  plates  can 
first  be  seen  on  the  second  or  third  day,       FIG.  118.— Colony 
then  appearing   as   transparent  grayish    of  the  bacillus    of 

«  .. ,  T  1        -•  r  mouse-septicemia:  X 

specks    with     irregular     borders,    from    8o  (FlUgge)> 
which  many  branched  processes  extend 
(Fig.   118).      Frankel  describes  them   as  resembling  in 
shape  the  familiar  branched  cells  occupying  the  lacunae 


428  PATHOGENIC  BACTERIA. 

of  bone.  When  further  developed  the  colonies  flow 
together  and  give  the  plate  a  cloudy  gray  appearance. 
The  gelatin  is  not  liquefied,  but  is  gradually  softened  and 
its  evaporation  thus  aided. 

In  gelatin  puncture-cultures  the  growth  is  quite  cha- 
racteristic, and  the  tendency  of  the  bacillus  to  grow 
anerobically  is  well  shown  (Fig.  119).  The  develop- 


FlG.   119. — Bacillus  of  mouse-septicemia:  gelatin  puncture-culture  three  and  a 
half  days  old  (Gunther). 

ment  takes  place  all  along  the  line  of  puncture,  but  is 
more  marked  below  than  at  the  surface.  The  growth 
takes  place  in  a  peculiar  form,  resembling  superimposed 
disks,  each  disk  separate  from  its  neighbors  and  consist- 
ing of  an  area  of  clouded  grayish  gelatin  reaching  almost 
to  the  walls  of  the  tube.  This  growth  develops  slowly, 
and  causes  a  softening  rather  than  an  actual  liquefaction 
of  the  gelatin. 

Upon  agar-agar  and  blood-serum  a  very  delicate,  trans- 
parent grayish  line  develops  along  the  path  of  the  needle. 
It  does  not  grow  upon  potato. 

The  bacillus  grows  at  the  room  temperature,  but  much 
better  at  the  temperature  of  the  incubator. 

The  disease  affects  quite  a  variety  of  animals,  notably 
hogs,  rabbits,  mice,  white  rats,  pigeons,  and  sparrows. 


MOUSE-SEPTICEMIA.  429 

The  guinea-pig,  which  is  generally  the  victim  of  labora- 
tory experiments,  is  not  susceptible  to  it.  Field-  and 
wood-mice,  cattle,  horses  asses,  dogs,  cats,  chickens,  and 
geese  are  immune. 

When  mice  are  inoculated  with  a  pure  culture  they 
soon  become  ill,  lose  their  appetite,  mope  in  a  corner, 
and  are  not  readily  disturbed.  As  the  disease  becomes 
worse  they  assume  a  sitting  posture  with  the  back  much 
bent;  the  eyelids  are  glued  together  by  adhesive  pus;  and 
when  death  comes  to  their  relief,  in  the  course  of  forty 
to  sixty  hours  after  inoculation,  they  remain  sitting  in 
the  same  characteristic  position. 

When  the  ears  of  rabbits  are  inoculated  with  the 
bacillus  from  cases  of  erysipelas  suis,  a  violent  inflam- 
matory edema  and  distinct  redness  occurs,  much  re- 
sembling erysipelas.  This  lesion  gradually  spreads,  in- 
volves the  head,  then  the  body  of  the  animal,  and  ulti- 
mately causes  death. 

When  swine  are  affected,  they  are  dull  and  weak,  and 
have  a  kind  of  paralytic  weakness  of  the  hind  quarters. 
The  temperature  is  elevated  ;  red  patches  appear  upon 
the  skin  and  swell  and  become  tender.  Death  follows  in 
two  or  three  days.  Sixty  per  cent,  of  the  diseased 
animals  die. 

In  all  animals  the  anatomical  changes  are  much  alike. 
The  disease  proves  to  be  a  septicemia,  and  the  bacilli  can 
be  found  in  all  the  organs,  especially  the  lungs  and  spleen. 
They  are  few  in  number  in  the  streaming  blood. 

As  the  organisms  stain  well  by  Gram's  method,  this 
stain  is  of  great  value  for  their  discovery  in  the  tissues, 
and  can  be  highly  recommended. 

Most  of  the  bacilli  occupy  the  capillary  blood-vessels  ; 
many  of  them  are  enclosed  in  leucocytes.  The  organs  in 
such  cases  do  not  appear  distinctly  abnormal,  except  the 
spleen,  which  is  considerably  enlarged.  The  mesenteric 
and  other  lymphatics  are  also  enlarged,  and  the  gastric 
and  intestinal  mucous  membranes  are  usually  inflamed 
and  mottled.  The  bacilli  also  occupy  the  intestinal  con- 


430  PATHOGENIC  BACTERIA. 

tents,  and  Kitt,  who  discovered  them  in  this  position, 
points  out  that  the  infection  of  swine  probably  takes 
place  by  the  entrance,  along  with  the  food,  of  the  fecal 
matter  of  diseased  animals  into  the  alimentary  apparatus 
of  others. 

Pasteur,  Chamberland,  Roux,  and  others  have  worked 
upon  a  protective  vaccination  based  upon  the  attenuation 
of  the  virulence  of  the  organism  by  passing  it  through 
rabbits.  Two  vaccinations  are  said  to  be  necessary  to 
produce  immunity.  The  vaccinated  animals,  however, 
may  be  a  source  of  infection  to  others,  and  should  always 
be  isolated.  Klemperer  in  1892  found  that  the  blood- 
serum  of  immunized  rabbits  would  save  infected  mice 
into  which  it  was  injected. 

Lorenz  in  1894  found  an  antitoxic  substance  in  the 
blood  of  rabbits  immunized  to  the  disease.  The  effect 
of  its  injection  into  other  animals  is,  however,  only  a 
temporary  immunity.  Later1  he  found  it  possible  to 
protect  hogs  against  the  disease  by  injecting  them  first 
with  a  serum  obtained  from  a  hog  immunized  in  the 
ordinary  manner  described  by  Pasteur,  afterward  with 
a  feeble  culture  of  the  bacillus,  and  finally  with  viru- 
lent cultures.  The  strength  of  the  serum  should  be 
determined  by  injecting  varying  quantities  of  it  into 
mice  infected  with  definite  amounts  of  a  culture  of 
known  virulence.  The  immunity  produced  by  Lorenz 
lasted  for  a  year. 

1  Centralbl.f.  Bakt.  u.  Parasitenk.,  Jan.,  1896,  p.  168. 


CHAPTER  X. 
RELAPSING   FEVER. 

As  long  ago  as  1873,  Obermeier  discovered  that  a 
flexible  spiral  organism,  about  o.  i  fj.  in  diameter  and 
from  20-40  fj.  in  length,  could  be  observed  in  the  blood 
of  patients  suffering  from  relapsing  fever. 

Although  many  of  the  best  bacteriologists  of  our  day 
have  occupied  themselves  with  the  study  of  this  spiril- 
lum, we  really  have,  at  present,  very  little  more  know- 
ledge than  that  given  us  by  Obermeier. 


FIG.  1 20. — Spirochgeta  febris  recurrentis;    x  650  (Heim). 

The  spirilla  (Fig.  120)  are  generally  very  numerous, 
are  long,  slender,  and  flexible  (spirochseta),  and  possess 
a  vigorous  movement  by  flagella.  The  ends  are  rather 
pointed. 

The  spirillum  stains  well  by  ordinary  methods,  but 
not  by  Gram's  method.  It  seems  to  be  a  strict  parasite, 
and  has  never  been  cultivated  artificially. 

Of  the  pathogenesis  of  the  organism  there  can  be  no 
doubt,  as  it  is  invariably  present  in  relapsing  fever  and 

431 


432  PA  THOGENIC  BA  CTERIA. 

undergoes  a  peculiar  cycle  of  changes  according  to  the 
stage  of  the  disease.  During  the  pyrexia  the  organisms 
are  found  in  the  blood  in  active  movement,  swimming 
both  by  rotation  on  the  long  axis  and  by  undulation. 
As  soon  as  the  crisis  comes  on  they  are  found  to  be  with- 
out motion,  most  of  them  enclosed  in  leucocytes  and 
seemingly  dead.  The  recurrence  of  the  paroxysm  has 
suggested  to  many  that  spores  are  formed  in  the  spiril- 
lum, but  no  one  has  been  successful  in  proving  that  this 
is  the  case.  Koch,  Carter,  and  Soudakewitch  have  all 
succeeded  in  giving  the  disease  to  monkeys,  and  Munch 
and  Moczutkowsky  have  gone  further  and  have  produced 
it  in  men  by  introducing  into  them  blood  from  diseased 
patients. 

Soudakewitch  finds  that   the  removal  of  the  spleen 
causes  the  disease  to  terminate  fatally  in  monkeys. 


CHAPTER    XI. 
BUBONIC   PLAGUE. 

THE  bacillus  of  bubonic  plague  (Fig.   121)  seems  to 
have   met   an   independent   discovery   at   the  hands   of 


FIG.  121. — Bacillus  of  bubonic  plague  (Yersin). 

Yersin  and  Kitasato  in  the  summer  of  1894,  during  the 
activity  of  the  plague  then  raging  at  Hong-Kong.  There 
seems  to  be  but  little  doubt  that  the  micro-organisms 
described  by  the  two  observers  are  identical. 

In  a  recent  study  of  the  plague,  Ogata1  states  that 
while  Kitasato  found  his  bacillus  in  the  blood  of  cadavers, 
Yersin  seldom  found  his  bacillus  in  the  blood,  but  always 
in  the  enlarged  lymphatic  glands.  Kitasato' s-  bacillus 
retains  the  color  when  stained  by  Gram's  method;  Yer- 
sin's  does  not.  Kitasato' s  bacillus  is  motile;  Yersin' s, 
non-motile.  The  colonies  of  Kitasato' s  bacillus  when 
grown  upon  agar  are  round,  irregular,  grayish-white  with 

1  Centralbl.  f,  Bakt.  u.  Parasitenk.,  Bd.  xxi.,  Nos.  20  and  21,  June  24, 
1897. 

28  433 


434 


PATHOGENIC  BACTERIA. 


a  bluish  tint,  and  resemble  glass-wool  when  slightly 
magnified;  Yersin's  bacillus  forms  white,  transparent  col- 
onies with  iridescent  edges.  Ogata,  in  the  investigation 


FIG.  122. — Bacilli  of  plague  and  phagocytes;    x  800.     From  human  lymphatic 
gland  (Aoyama). 

of  the  cases  that  came  into  his  hands  found  a  bacillus 
that  resembles  that  of  Yersin,  but  not  that  of  Kitasato. 

The  bubonic  plague  is  an  extremely  fatal  infectious 
disease,  whose  ravages  in  the  hospital  in  which  Yersin 
made  his  observations  carried  off  95  per  cent,  of  the 
cases.  It  affects  both  men  and  animals,  and  is  character- 
ized by  sudden  onset,  high  fever,  prostration,  delirium, 
and  the  occurrence  of  lymphatic  swellings — buboes — 
affecting  chiefly  the  inguinal  glands,  though  not  infre- 
quently the  axillary,  and  sometimes  the  cervical,  glands. 
Death  comes  on  in  severe  cases  in  forty-eight  hours.  If 
the  case  is  of  longer  duration,  the  prognosis  is  said  to  be 
better.  Autopsy  in  fatal  cases  reveals  the  characteristic 
enlargement  of  the  lymphatic  glands,  whose  contents  are 
soft  and  sometimes  purulent. 

Wyssokowitz  and  Zabolotmy1  describe  two  forms  of 
the  disease: 

1  Ann.  de  rinst.  Pasteur,  Aug.  25,  1897,  xi.,  8,  p.  665. 


BUBONIC  PLAGUE.  435 

1.  Plague  with  buboes. 

2.  Plague  without  buboes,  but  with  a  primary  specific 
pneumonia  in  which  the  bacilli  occur  in  immense  num- 
bers in  the  affected  pulmonary  tissue,  but  sparingly  in  the 
blood  and  kidney. 

The  studies  of  Kitasato  and  Yersin  show  that  in  blood 
drawn  from  the  finger-tips  and  in  the  softened  contents 
of  the  glands  a  small  bacillus  is  demonstrable.  The 
organisms  are  small,  stain  much  more  distinctly  at  the 
ends  than  in  the  middle,  so  that  they  resemble  diplo- 
cocci,  and  in  fresh  specimens  seem  to  be  surrounded  by 
a  capsule.  Kitasato  compares  the  organism  to  the  well- 
known  bacillus  of  ckicken-cholera.  It  is  feebly  motile 
(according  to  Abel,  entirely  non-motile),  and  does  not 
seem  to  form  spores.  Nothing  is  said  in  the  original 
descriptions  about  the  presence  of  flagella,  though  it  is 
probable  from  the  studies  of  Gordon  l  that  some,  at  least, 
of  the  bacilli  may  be  possessed  of  them.  It  does  not 
stain  by  Gram's  method. 

When  cultures  are  made  from  the  softened  contents  of 
the  buboes  the  bacillus  may  be  obtained  almost  or  quite 
pure,  and  is  found  to  develop  upon  artificial  culture- 
media.  In  bouillon  a  diffuse  cloudiness  results  from 
the  growth,  as  observed  by  Kitasato,  though  in  Yersin's 
observations  the  culture  more  nearly  resembled  erysipe- 
las cocci,  and  contained  zooglea  attached  to  the  sides  and 
at  the  bottom  of  the  tube  of  nearly  clear  fluid. 

According  to  Haffkine,2  when  an  inoculated  bouillon 
culture  is  allowed  to  stand,  perfectly  at  rest,  on  a  solid 
shelf  or  table  a  characteristic  appearance  results.  In 
from  twenty-four  to  forty-eight  hours,  the  liquid  remain- 
ing limpid,  flakes  appear  underneath  the  surface,  forming 
little  islands  of  growth,  which  in  the  next  twenty-four 
to  forty-eight  hours  grow  down  into  a  long  stalactite-like 
jungle,  the  liquid  always  remaining  clear.  In  four  to 

1  Centralbl.  f.  Bakt.  n.  Parasitenk.,  Sept.  6,  1897,  Bd.  xxii.,  Nos.  6  and  7, 
p.  170. 

2  Brit.  Med.  Jour.,  June  12,  1897,  p.  1461. 


436  PA  THOGENIC  BA  CTERIA . 

six  days  the  islands  are  still  more  compact  and  solidified. 
If  the  vessel  be  disturbed,  the  islands  fall  like  snow  and 
are  deposited  at  the  bottom. 

Upon  gelatin  plates  at  22°  C.  the  colonies  may  be  ob- 
served in  twenty-four  hours  by  the  naked  eye.  They  are 
pure  white  or  yellowish-white,  spherical  in  the  deep  gela- 
tin, flat  upon  the  surface,  and  are  about  the  size  of  a 
pin's  head.  The  gelatin  is  not  liquefied.  The  borders 
of  the  colonies  are,  upon  microscopic  examination,  found 
to  be  sharply  defined  and  to  become  more  granular  as 
their  age  increases.  The  superficial  colonies  occasionally 
are  surrounded  by  a  fine,  semi-transparent  zone. 

In  gelatin  puncture-cultures  the  development  is  scant. 
The  medium  is  not  liquefied  (?);  the  growth  takes  place 
in  the  form  of  a  fine  duct,  little  points  being  seen  on  the 
surface  and  in  the  line  of  puncture. 

Upon  agar-agar — glycerin  agar-agar  is  best — the  bacilli 
grow  freely,  the  colonies  being  whitish  in  color,  with  a 
bluish  tint  by  reflected  light.  Under  the  microscope 
they  appear  moist,  with  rounded,  uneven  edges.  The 
small  colonies  are  said  to  resemble  little  tufts  of  glass- 
wool;  the  larger  ones  have  large  round  centers.  Micro- 
scopic examination  of  the  bacilli  grown  upon  agar-agar 
reveals  the  presence  of  long  chains  resembling  strepto- 
cocci. 

Klein1  states  that  the  colonies  develop  quite  readily 
upon  gelatin  made  from  beef-bouillon  (not  infusion), 
appearing  in  twenty-four  hours,  at  20°  C.,  as  small,  gray, 
irregularly  rounded  dots.  Magnification  shows  the  col- 
onies to  be  serrated  at  the  edges  and  made  up  of  short, 
oval,  sometimes  double  bacilli.  Some  colonies  contrast 
markedly  with  their  neighbors  in  that  they  are  large, 
round,  or  oval,  and  consist  of  longer  or  shorter,  straight 
or  looped  threads  of  bacilli.  The  appearance  was  much 
like  that  of  the  young  colonies  of  the  Proteus  vulgaris. 
At  first  Klein  regarded  these  as  contaminations,  but  later 
he  was  led  to  believe  that  their  occurrence  was  character- 

1  Ccntralbl.f.  Bakt.  u.  Pamsittnk.,  xxi.,  Nos.  24  and  25,  July  10,  1897. 


BUBONIC  PLAGUE.  437 

istic  of  the  plague  bacillus.  The  peculiarities  of  these 
colonies  cannot  be  recognized  after  forty-eight  hours. 

Involution-forms  on  partly  desiccated  agar-agar  not  con- 
taining glycerin  are  said  by  Haff  kine  to  be  characteristic. 
The  microbes  swell  up  and  form  large,  round,  oval,  pea- 
or  spindle-shaped  or  biscuit-like  bodies,  which  may  attain 
twenty  times  the  normal  size  and  in  growing  gradually 
lose  the  ability  to  take  up  the  stain.  Such  involution- 
forms  are  not  seen  in  liquid  culture. 

Hankin  and  Leumann  l  recommend  for  the  differential 
diagnosis  of  the  plague  bacillus  the  addition  of  2.5-3.5 
per  cent,  of  salt  to  the  agar-agar.  When  transplanted 
from  ordinary  agar-agar  to  the  salt  agar-agar  the  involu- 
tion-forms which  are  so  characteristic  of  the  plague  ba- 
cillus form  with  exceptional  rapidity. 

Upon  blood-serum  the  growth  at  the  temperature  of 
the  incubator  is  luxuriant.  It  forms  a  moist  layer  of  a 
yellowish-gray  color,  and  is  unaccompanied  by  liquefac- 
tion of  the  serum. 

Upon  potato  no  growth  occurs  at  ordinary  tempera- 
tures. When  the  potato  is  stood  away  for  a  few  days  in 
the  incubator  a  scanty,  dry,  whitish  layer  develops. 

Abel  found  the  best  culture-medium  to  be  2  per  cent, 
alkaline  pepton  solution  with  i  or  2  per  cent,  of  gelatin, 
as  recommended  by  Yersin  and  Wilson. 

The  bacillus  develops  under  conditions  of  aerobiosis  and 
anaerobiosis.  In  glucose-containing  media  it  does  not 
form  gas.  No  indol  is  formed.  Ordinarily  the  culture- 
medium  is  acidified  by  the  development  of  an  acid  that 
persists  for  three  weeks  or  more. 

By  frequent  passage  through  animals  of  the  same 
species  the  bacillus  increases  very  much  in  virulence. 
Curiously  enough,  however,  the  observations  of  Knorr, 
substantiated  by  Yersin,  Caltnette  and  Borrel,  show  that 
the  bacillus  made  virulent  by  frequent  passage  through 
mice  is  not  increased  in  virulence  for  rabbits.2 

1  Centralbl.  f.  Bakt.  u.  Parasitenk.,  Oct.,  1897,  Bd.  xxii.,  Nos.  16  and  17, 
p.  438.  2  Ann.  de  /' Inst.  Pasteur,  July,  1895. 


438  PATHOGENIC  BACTERIA. 

Kitasato  found  that  mice,  rats,  guinea-pigs,  and  rabbits 
are  all  susceptible;  pigeons  are  immune.  Julian  Haw- 
thorne, in  his  paper  in  the  Cosmopolitan,  speaks  of  hav- 
ing seen  cats  and  dogs  dying  of  the  disease,  but  no  men- 
tion is  made  of  these  animals  in  the  scientific  papers  I 
have  read.  When  blood,  lymphatic  pulp,  or  pure  cul- 
tures are  inoculated  into  them,  the  animals  become  ill  in 
from  one  to  two  days,  according  to  their  size.  Their  eyes 
become  watery,  they  begin  to  show  disinclination  to  take 
food  or  to  make  any  bodily  effort,  the  temperature  rises 
to  41.5°  C.,  they  remain  quietly  in  a  corner  of  the  cage, 
and  die  with  convulsive  symptoms  in  from  two  to  five 
days. 

Devell *  has  found  that  frogs  are  susceptible  to  the  dis- 
ease. 

Wyssokowitz  and  Zabolotmy2  found  monkeys  to  be 
highly  susceptible  to  plague,  especially  when  inoculated 
subcutaneously.  When  so  small  an  inoculation  was 
made  as  a  puncture  with  a  pin  dipped  in  a  culture  of  the 
bacillus,  the  puncture  being  made  in  the  palm  of  the 
hand  or  sole  of  the  foot,  the  monkeys  always  died  in 
from  three  to  seven  days.  In  these  cases  the  local  edema 
observed  by  Yersin  did  not  occur.  They  point  out  the 
interest  attaching  to  infection  through  so  insignificant  a 
wound  and  without  local  lesions. 

According  to  Yersin,  an  infiltration  or  watery  edema 
can  be  observed  in  a  few  hours  about  the  point  of  inocula- 
tion. The  autopsy  shows  the  infiltration  to  be  made  up 
of  a  yellowish  gelatinous  exudation.  The  spleen  and 
liver  are  enlarged,  the  former  often  presenting  an  appear- 
ance much  like  an  eruption  of  iniliary  tubercles.  Some- 
times there  is  universal  swelling  of  the  lymphatic  glands. 
Bacilli  are  found  in  the  blood  and  in  all  the  internal 
organs.  Very  often  there  are  eruptions  during  life,  and 
upon  the  inner  abdominal  walls  there  are  petechiae  and 
occasional  hemorrhages.  The  intestine  is  hyperemic,  the 

1  Ctntralbl.f.  Rakt.  u.  Parasitenk.,  Oct.  12,  1897. 

*  Ann.  df  r  fnst.  Pasteur,  Aug.  25,  1897,  xi.,  8,  p.  665. 


BUBONIC  PLAGUE.  439 

adrenals  congested.  There  are  often  sero-sanguinolent 
effusions  into  the  serous  cavities. 

Klein  l  states  that  the  intraperitoneal  injection  of  the 
bacillus  into  guinea-pigs  is  of  diagnostic  value,  produc- 
ing in  twenty-four  to  forty-eight  hours  a  thick  cloudy 
peritoneal  exudate  rich  in  leukocytes  and  containing 
characteristic  chains  of  the  plague  bacillus. 

Animals  fed  upon  cultures  or  upon  the  flesh  of  other 
animals  dead  of  the  disease  became  ill  and  died  with 
typical  symptoms.  When  Klein  inoculated  animals  with 
the  dust  of  dwelling-houses  in  which  the  disease  had 
occurred,  some  died  of  tetanus,  one  from  plague.  Many 
rats  and  mice  in  which  examination  showed  the  charac- 
teristic bacilli  died  spontaneously  in  Hong-Kong. 

Yersiii  showed  that  flies  also  die  of  the  disease.  Mace- 
rating and  crushing  a  fly  in  bouillon,  he  not  only  suc- 
ceeded in  obtaining  the  bacillus  from  the  medium,  but 
infected  an  animal  with  it. 

Nuttall,2  in  reviewing  Yersin's  fly-experiment,  found 
the  statement  true,  and  showed  that  flies  fed  with  the 
cadavers  of  plague-infected  mice  died  in  a  variable 
length  of  time.  Large  numbers  of  plague  bacilli  were 
found  in  their  intestines.  He  also  found  that  bed-bugs 
allowed  to  prey  upon  infected  animals  took  up  large 
numbers  of  the  plague  bacilli  and  retained  them  for  a 
number  of  days.  These  bugs  did  not,  however,  infect 
healthy  animals  when  allowed,  subsequently,  to  feed 
upon  them.  Nuttall  is  not,  however,  satisfied  that  the 
number  of  his  experiments  upon  this  point  was  great 
enough  to  be  conclusive. 

Ogata  found  that  the  plague  bacillus  existed  in  the 
bodies  of  fleas  found  upon  diseased  rats.  One  of  these 
he  crushed  between  sterile  object-glasses  and  introduced 
into  the  subcutaneous  tissues  of  a  mouse,  which  died 
in  three  days  with  typical  lesions  of  the  plague,  a  con- 
trol-animal remaining  well.  Some  guinea-pigs  taken 

1  Centralbl.  f.  Bakt.  u.  Parasitenk.,  xxi.,  No.  24,  July  10,  1897,  p.  849. 

2  Ibid.,  Aug.  13,  1897. 


440  PATHOGENIC  BACTERIA. 

for  experimental  purposes  into  a  plague  district,  and 
kept  carefully  isolated,  died  spontaneously  of  the  disease, 
presumably  because  of  insect  infection. 

Yersin  found  that  when  cultivated  for  any  length  of 
time  upon  culture-media,  especially  agar-agar,  the  viru- 
lence was  rapidly  lost  and  the  bacillus  eventually  died. 
On  the  other  hand,  when  constantly  inoculated  from 
animal  to  animal  the  virulence  of  the  bacillus  is  much 
increased. 

The  bacillus  probably  attenuates  readily.  Kitasato 
found  that  it  did  not  seem  able  to  withstand  desicca- 
tion longer  than  four  days ;  and  Yersin  found  that  al- 
though it  could  be  secured  from  the  soil  beneath  an 
infected  house  at  a  depth  of  4-5  c.cm.,  the  virulence 
of  such  bacilli  was  lost. 

Kitasato  found  that  the  bacillus  was  killed  by  two 
hours'  exposure  to  0.5  per  cent,  carbolic  acid,  and  also 
by  exposure  to  a  temperature  of  80°  C.  Ogata  found 
that  the  bacillus  was  instantly  killed  by  5  per  cent,  car- 
bolic acid,  and  in  fifteen  minutes  by  0.5  per  cent,  carbolic 
acid.  In  o.  i  per  cent,  sublimate  solution  it  is  killed  in 
five  minutes. 

It  seems  possible  to  make  a  diagnosis  of  the  disease  in 
doubtful  cases  by  examining  the  blood,  but  it  is  admitted 
that  a  good  deal  of  bacteriologic  practice  is  necessary  for 
the  purpose. 

Abel  finds  that  the  blood  may  yield  fallacious  results 
because  of  the  rather  variable  appearance  of  the  bacilli, 
which  are  sometimes  long  and  easily  mistaken  for  other 
bacteria.  He  deems  the  best  tests  to  be  the  inoculation 
of  broth-cultures  and  subsequent  inoculation  into  ani- 
mals, which  he  advises  should  have  been  previously 
vaccinated  against  the  streptococcus.  Plague  bacilli 
persist  in  the  urine  a  week  after  convalescence. 

Wilson,  of  the  Hoagland  Laboratory,  found  the  thermal 
death-point  of  the  organism  was  one  or  two  degrees 
higher  than  that  of  the  majority  of  pathogenic  bacteria 
of  the  non-sporulating  variety,  and  that,  unlike  cholera, 


BUBONIC  PLAGUE.  441 

the  influence  of  sunlight  and  desiccation  cannot  be  relied 
upon  to  limit  its  viability. 

Kitasato's  experiments  first  showed  that  it  is  possible 
to  bring  about  immunity  to  the  disease,  and  Yersin, 
working  in  India,  and  Fitzpatrick,  in  New  York,  have 
successfully  immunized  large  animals  (horses,  sheep, 
goats).  The  serum  of  these  immunized  animals  con- 
tains an  antitoxin  capable  not  only  of  preventing  the  dis- 
ease, but  also  of  curing  it  in  mice  and  guinea-pigs  and 
probably  in  man. 

HafFkine  in  his  experiments  followed  the  line  of  pre- 
ventive inoculation  as  employed  against  cholera.  Bouil- 
lon cultures  were  used  in  which  floating  drops  of  butter 
were  employed  to  make  the  islands  of  plague  bacilli 
float.  The  cultures  were  grown  for  a  month  or  so,  suc- 
cessive crops  of  the  island-stalactite  growth  as  it  formed 
having  been  precipitated  by  agitating  the  tube.  In  this 
manner  there  was  obtained  an  "intense  extra-cellular 
toxin"  containing  large  numbers  of  the  bacilli.  The 
culture  was  killed  by  exposure  to  a  temperature  of  70° 
C.  for  one  hour,  and  the  mixture  used  in  doses  of  about 
3  c.cm.  as  a  preventive  inoculation.  In  the  Byculla 
Gaol,  where  Haffkine's  experiments  numbered  over  one 
hundred,  a  decided  prophylactic  effect  was  observed  in 
twelve  to  fourteen  hours  in  men  already  advanced  in  the 
stage  of  incubation. 

Wyssokowitz  and  Zabolotmy,  whose  studies  have 
already  been  quoted,  used  96  monkeys  in  the  study  of 
the  value  of  the  "plague-serums,"  and  found  that 
when  the  treatment  is  begun  within  two  days  from  the 
time  of  inoculation  the  animals  can  be  saved,  even 
though  symptoms  of  the  disease  are  marked.  After  the 
second  day  the  treatment  cannot  be  relied  upon.  The 
dose  necessary  was  20  c.cm.  of  a  serum  having  a  potency 
of  i  :  10.  If  too  little  serum  was  given,  the  course  of 
the  disease  was  slowed,  the  animal  improved  for  a  time 
and  then  suffered  a  relapse,  and  died  in  from  thirteen  to 
seventeen  days.  The  serum  also  produced  immunity, 


442  PA  THOGENIC  BA  CTERIA. 

but  of  only  ten  to  fourteen  days'  duration.  Immunity 
lasting  three  weeks  was  conferred  by  inoculating  a  mon- 
key with  an  agar-agar  culture  heated  to  60°  C.  If  too 
large  a  dose  of  such  a  culture  was  given,  however,  the 
animal  was  enfeebled  and  remained  susceptible. 


CHAPTER    XII. 
TETRAGENUS. 

THERE  can  sometimes  be  found  in  the  normal  saliva, 
more  commonly  in  tuberculous  sputum,  and  still  more 
commonly  in  the  cavities  of  tuberculosis  pulmonalis,  a 
large  micrococcus  grouped  in  fours  and  known  as  the 
Micrococcus  tetragenus  (Fig.  123).  It  was  discovered  by 


FIG.  123. — Micrococcus  tetragenus  in  pus  from  a  white  mouse;  x  615  (Heim). 

Gaffky,  and  subsequently  carefully  studied  by  Koch  and 
Gaffky.  It  sometimes  occurs  in  the  pus  of  acute  ab- 
scesses, and  may  be  of  importance  in  connection  with 
the  pulmonary  abscesses  which  so  often  complicate  tu- 
berculosis. 

The  cocci  are  rather  large,  measuring  about  i  //  in 
diameter.  In  cultures  they  show  no  particular  arrange- 
ment among  themselves,  but  in  the  blood  and  tissues  of 
animals  they  commonly  appear  arranged  in  groups  of 
four  surrounded  by  a  transparent  gelatinous  capsule. 

The  organfem  stains  well  by  ordinary  methods,  and 

443 


444  PA  THOGENIC  BA CTERIA. 

most  beautifully  by  Gram's  method,  by  which  it  can  be 
best  demonstrated  in  tissues. 

Upon  gelatin  plates  small  white  colonies  are  produced 
in  from  twenty-four  to  forty-eight  hours.  Under  the 
microscope  they  are  found  to  be  spherical  or  elongate 
(lemon-shaped),  finely  granular,  and  lobulated  like  a 
raspberry  or  a  mulberry.  When  superficial  they  form 
white,  elevated,  rather  thick  masses  1-2  mm.  in  diameter 
(Fig.  124). 

In    gelatin    punctures  a   large  white  surface-growth 


FIG.  124. — Micrococcus  tetragenus:  colony  twenty-four  hours  old  upon  the  sur- 
face of  an  agar-agar  plate;  x  100  (Heim). 

takes  place,  but  very  scant  development  occurs  in  the 
puncture,  where  the  small  spherical  colonies  generally 
remain  isolated. 

Upon  the  surface  of  agar-agar  spherical  white  colonies 
are  produced.  They  may  remain  isolated  or  may  become 
confluent. 

Upon  potato  a  luxuriant  thick,  white  growth  occurs. 

The  growth  upon  blood-serum  is  also  abundant,  espe- 
cially at  the  temperature  of  the  incubator.  It  has  no 
distinctive  peculiarities. 

The  introduction  of  tuberculous  sputum  or  of  a  most 
minute  quantity  of  a  pure  culture  of  this  coccus  into 
white  mice  generally  causes  a  fatal  septicemia. 


TETRAGENUS.  445 

The  organisms  are  found  in  small  numbers  in  the 
heart's  blood,  but  arev  numerous  in  the  spleen,  lungs, 
liver,  and  kidneys. 

House-mice  and  field-mice  are  comparatively  immune  ; 
dogs  and  rabbits  are  also  highly  resistant.  Guinea-pigs 
sometimes  die  from  general  infection,  though  sometimes 
local  abscesses  may  be  the  only  result  of  subcutaneous 
inoculation. 

The  tetragenococci  are  of  no  special  importance  in 
human  pathology,  but  probably  hasten  the  tissue-necrosis 
in  tuberculosis  pulmonalis,  and  may  aid  in  the  formation 
of  abscesses  of  the  lung  and  contribute  to  the  production 
of  the  hectic  fever. 


CHAPTER   XIII. 
INFLUENZA. 

NOTWITHSTANDING  a  large  number  of  bacteriologic 
examinations  conducted  for  the  purpose  of  determining 
the  cause  of  influenza,  it  was  not  until  1892,  after  the 
great  epidemic,  that  there  was  found  simultaneously  by 
Canon  and  Pfeiffer  a  bacterium  which  conformed,  at  least 
in  large  part,  to  the  requirements  of  specificity. 

The  observers  mentioned  found  the  same  organism — 
one  in  the  blood  of  influenza  patients,  the  other  in  the 
purulent  bronchial  discharges. 

The  specific  organisms  (Fig.  125)  are  bacilli,  very  small 
in  size,  having  about  the  same  diameter  as  the  bacillus 


. 

:«*  -,'» 


- 


FIG.  125.  —  Bacillus  influenzae,  from  tt  gelatin  culture;    x   looo  (Itzerott  and 

Niemann). 

of  mouse-septicemia,  but  only  about  half  as  long  (0.2  by 
0.5  p).  They  are  usually  solitary,  but  may  be  united  in 
chains  of  three  or  four  elements.  They  stain  rather 

446 


INFLUENZA.  447 

poorly,  except  with  such  concentrated  penetrating  stains 
as  carbol-fuchsin  and  Loffler's  alkaline  methylene  blue, 
and  even  with  these  the  bacilli  stain  more  deeply  at  the 
ends  than  in  the  middle,  so  that  they  appear  not  a  little 
like  diplococci. 

For  the  demonstration  of  the  bacilli  in  the  blood  Canon 
recommends  a  rather  complicated  method.  The  blood  is 
spread  upon  clean  cover-glasses  in  the  usual  way,  thor- 
oughly dried,  and  then  fixed  by  immersion  in  absolute 
alcohol  for  five  minutes.  The  stain  which  seems  best  is 
Czenzynke's : 

Concentrated  aqueous  solution  of  methylene 

blue,  40 ; 

0.5  per  cent,  solution  of  eosin  in  70  per  cent. 

alcohol,  20 ; 

Distilled  water,  40. 

The  cover-glasses  are  immersed  in  this  solution,  and  kept 
in  the  incubator  for  three  to  six  hours,  after  which  they 
are  washed  in  water,  dried,  and  then  mounted  in  Canada 
balsam.  By  this  method  the  erythrocytes  are  stained  red, 
the  leucocytes  blue,  and  the  bacillus,  which  is  also  blue, 
appears  as  a  short  rod  or  often  as  a  dumb-bell. 

Sometimes  large  numbers  of  the  bacilli  are  present ; 
sometimes  very  few  can  be  found  after  prolonged  search. 
They  are  often  enclosed  within  the  leucocytes.  It  really 
is  not  necessary  to  pursue  so  tedious  a  staining  method 
for  demonstrating  the  bacilli,  for  they  stain  quite  well  by 
ordinary  methods.  They  do  not  stain  by  Gram's  method. 

The  bacillus  is  non-motile,  and,  so  far  as  is  known, 
does  not  form  spores.  Its  resisting  powers  are  very  re- 
stricted, as  it  speedily  succumbs  to  drying,  and  is  cer- 
tainly killed  by  an  exposure  to  a  temperature  of  60°  C. 
for  five  minutes.  It  will  not  grow  at  any  temperature 
below  28°  C. 

The  bacillus  does  not  grow  in  gelatin  or  upon  ordinary 
agar-agar.  Upon  glycerin  agar-agar,  after  twenty-four 
hours  in  the  incubator,  minute  colorless,  transparent, 


448  PATHOGENIC  BACTERIA. 

drop-like  cultures  may  be  seen  along  the  line  of  inocula- 
tion. They  do  not  look  unlike  condensed  moisture,  and 
Kitasato  makes  a  special  point  of  the  fact  that  the  colo- 
nies never  become  confluent.  The  colonies  may  at  times 
be  so  small  as  to  require  a  lens  for  their  discovery. 

In  bouillon  a  scant  development  occurs,  small  whitish 
particles  appearing  upon  the  surface,  subsequently  sink- 
ing to  the  bottom  and  causing  a  "woolly"  deposit  there. 
While  the  growth  is  so  delicate  in  these  ordinary  media, 
the  bacillus  grows  quite  well  upon  culture-media  contain- 


FIG.  126. — Bacillus  of  influenza;  colonies  on  blood  agar-agar;  low  magnifying 
power  (Pfeiffer). 

ing  hemoglobin  or  blood,  and  can  be  transferred  from 
culture  to  culture  many  times  before  it  loses  its  vitality. 
It  cannot  be  positively  proven  that  this  bacillus  is  the 
cause  of  influenza,  but  from  the  fact  that  the  bacillus 
can  be  found  only  in  cases  of  influenza,  that  its  presence 
corresponds  with  the  course  of  the  disease  in  that  it  is 
present  as  long  as  the  purulent  secretions  last,  and  then 
disappears,  and  that  Pfeiffer  was  able  to  demonstrate  its 
presence  in  all  cases  of  uncomplicated  influenza,  his  con- 
clusion that  the  bacillus  is  specific  is  certainly  justifiable. 


INFLUENZA.  449 

The  bacillus  is  pathogenic  for  certain  of  the  laboratory 
animals,  the  guinea-pig  in  particular  being  subject  to 
fatal  infection.  The  dose  required  to  cause  death  of  a 
guinea-pig  varies  considerably,  in  the  immunization  ex- 
periments of  Deline  and  Kole1  ^V  of  a  24-hour  old  culture 
being  fatal  in  twenty-four  hours.  These  scholars  found 
that  the  toxicity  of  the  culture  resides  not  in  a  soluble 
toxin,  but  in  the  bodies  of  the  bacilli.  The  outcome  of 
the  researches,  which  were  made  most  scientifically  and 


FIG.  127. — bacillus  of  influenza;  cover-glass  preparation  of  sputum  from  a  case 
of  influenza,  showing  the  bacilli  in  leukocytes;  highly  magnified  (Pfeiflfer). 

painstakingly,  was  the  total  failure  to  produce  immunity. 
Increasing  doses  of  the  cultures  injected  into  the  peri- 
toneum resulted  in  enabling  the  animals  to  resist  rather 
more  than  a  fatal  dose,  but  never  enabled  them  to  main- 
tain vitality  when  large  doses  were  administered.  This 
discovery  is  in  exact  harmony  with  the  familiar  clinical 
observation  that,  instead  of  an  individual  being  immune 
after  an  attack  of  influenza,  he  is  as  susceptible  as  before, 
if  not  more  so. 

1  Zeitschrift  fur  Hygiene,  etc.,  Bd.  xxiv.,  1897,  Heft.  2. 
29 


450  PATHOGENIC  BACTERIA. 

A.  Catanni,  Jr.1  trephined  rabbits  and  injected  influ- 
enza toxin  into  their  brains,  at  the  same  time  trephining 
control-animals,  into  some  of  whose  brains  he  injected 
water.  The  results  were  that  animals  thus  receiving 
0.5-1  mgr.  of  the  living  culture  constantly  died  in 
twenty-four  hours  with  all  the  nervous  symptoms  of  the 
disease,  dyspnea,  paralysis  beginning  in  the  posterior 
extremities  and  extending  over  the  whole  body,  clonic 
convulsions,  stiffness  of  the  neck,  etc.  Control-animals 
injected  with  a  variety  of  pathogenic  bacteria  in  the 
same  manner  never  manifested  similar  symptoms.  The 
virulence  of  the  bacillus  was  also  observed  to  increase 
rapidly  when  transplanted  from  brain  to  brain. 

1  Zeitschrift  fitr  Hygiene,  etc.,  Bd.  xxiii.,  1896. 


CHAPTER   XIV. 
MEASLES. 

IN  1892,  Canon  and  Pielicke,  after  the  investigation  of 
fourteen  cases  of  measles,  reported  the  discovery  of  a 
specific  bacillus  in  the  blood  in  that  disease. 

The  organism  is  quite  variable  in  size,  sometimes 
being  quite  small  and  resembling  a  diplococcus,  some- 
times larger,  and  occasionally  quite  long,  so  that  one 
bacillus  may  be  as  long  as  the  diameter  of  a  red  blood- 
corpuscle. 

The  discovery  was  made  by  means  of  a  peculiar  method 
of  staining,  as  follows :  The  blood  is  spread  in  a  very 
thin,  even  layer  upon  perfectly  clean  cover-glasses,  and 
fixed  by  five  to  ten  minutes'  immersion  in  absolute  alco- 
hol. These  glasses  are  then  placed  in  a  stain  consisting 
of 

Concentrated  aqueous  solution  of  methylene  blue,  40 ; 
0.25  per  ct.  solution  of  eosin  in  70  per  ct.  alcohol,  20  ; 
Distilled  water,  40, 

and  stood  in  the  incubator  at  37°  C.  for  from  six  to 
twenty-four  hours.  The  bacilli  do  not  all  stain  uni- 
formly. 

The  discoverers  of  the  bacillus  claim  to  have  made  it 
grow  several  times  in  bouillon,  but  failed  to  induce  a 
growth  upon  other  media. 

The  bacilli  do  not  stain  by  Gram's  method  ;  they  seem 
to  have  motility ;  no  spores  were  observed.  They  were 
found  not  only  in  the  blood,  but  also  in  the  secretions 
from  the  nose  and  eyes.  They  are  said  to  persist  through- 
out the  whole  course  of  the  disease,  even  occasionally 
being  found  after  the  fever  subsides. 

451 


452  PATHOGENIC  BACTERIA. 

Czajrowski  asserts  that  the  bacillus  can  be  cultivated 
upon  various  albuminous  media  except  gelatin  and  agar. 
On  glycerin  agar-agar,  especially  with  the  addition  of 
hematogen,  and  on  blood-serum,  they  should  grow  in 
three  or  four  days  with  an  appearance  like  that  of  dew- 
drops.  Under  the  microscope  the  colonies  are  structure- 
less. Mice  die  of  a  septicemia  after  a  subcutaneous  in- 
oculation. 

An  interesting  field  for  experimentation  has  been 
opened  by  Behla,1  who  seems  to  have  successfully  inocu- 
lated a  sucking-pig  with  measles  by  introducing  some  of 
the  nasal  secretion  from  a  case  of  measles  into  the  nose, 
which  had  been  prepared  to  receive  it  by  scratching  with 
a  wire. 

1  Centralbl.f.  Bakt.  ti.  Parasitenk.,  Oct.  24,  1896,  Bd.  xx.,  Nos.  16  and  17, 
p.  36. 


D.    MISCELLANEOUS. 


CHAPTER   I. 
SYMPTOMATIC  ANTHRAX. 

"SYMPTOMATIC  ANTHRAX,"  charbon  symptomatique, 
Rauschbrand,  "quarter-evil,"  and  u black-leg"  are  the 
various  names  applied  to  a  peculiar  disease  of  cattle  com- 
mon during  the  summer  season  in  the  Bavarian  Alps, 
Baden,  Schleswig-Holstein,  and  some  parts  of  the  United 
States,  characterized  by  the  occurrence  of  irregular,  em- 
physematous,  crepitating  subcutaneous  pustules.  Dis- 
eased areas  are  also  found  in  the  muscles,  and  are  most 
common  over  the  quarters,  hence  the  name  "qxiarter- 
evil."  When  incised  the  affected  tissues  have  a  dark 
color  and  contain  a  dark,  bloody  serum. 

The  micro-organismal  nature  of  the  disease  had  been 
suspected  from  an  early  date,  but  until  the  work  of 
Faser  and  Bellinger  the  disease  was  confounded  with 
anthrax.  Still  later,  Arloing,  Thomas,  Cornevin,  and 
Kitasato  studied  the  disease,  and  succeeded  in  demon- 
strating the  specific  micro-organism,  which  Kitasato 
successfully  cultivated  upon  artificial  media. 

The  bacillus  which  the  results  of  these  labors  brought 
to  light  is  a  rather  large  individual  (3-5  n  in  length, 
0.5-0.6  /j-  in  breadth)  with  rounded  ends.  The  bacilli 
are  occasionally  united  in  twos,  but  are  never  united  in 
long  chains  (Fig.  128).  They  are  actively  motile  (Thoinot 
and  Masselin  say  scarcely  at  all  motile)  when  examined 
in  the  hanging  drop,  but  after  a  short  time,  perhaps 
because  of  the  exposure  to  the  oxygen  required  in  the 
hanging-drop  preparation,  the  movement  is  lost  and  the 
bacilli  die.  When  stained  by  Loffler's  method  a  con- 
siderable number  of  flagella  can  be  demonstrated.  L,arge 

453 


454  ?A  THOGENIC  BA  CTERIA . 

oval  spores  are  found  ;  by  their  presence  they  distort  the 
bacilli  in  which  they  occur,  causing  them  to  assume  a 
spindle  shape  (clostridium),  or,  when  two  are  united  and 
a  spore  occupies  one  of  them,  a  drumstick  shape.  In- 


J 

'    >  > 

^^  ** 


*s* 

m 


o         X 


FIG.  128. — Bacillus  of  symptomatic  anthrax,  containing  spores,  from  an  agar- 
agar  culture;    x  1000  (Frankel  and  Pfeiffer). 

volution-forms  are  exceedingly  common  in  old  cul- 
tures, and  are  of  enormous  size  and  of  granular  appear- 
ance. 

The  bacillus  can  be  stained  with  the  ordinary  aqueous 
solutions  of  the  anilin  dyes,  but  will  not  retain  the  color 
by  Gram's  method  or  Weigert's  fibrin  method.  It  can 
be  colored  in  sections  of  tissue  with  Loffler's  solution, 
and  can  be  observed  in  the  blood  without  staining  shortly 
after  death. 

The  spores,  which  can  be  stained  by  ordinary  methods, 
are  quite  resistant  to  the  action  of  heat  and  disinfect- 
ants, and  withstand  the  effects  of  drying  for  a  consider- 
able length  of  time. 

The  bacillus  of  symptomatic  anthrax  (Fig.  129)  is  a 
strictly  anaerobic,  parasitic  bacterium.  It  grows  at  tem- 
peratures above  18°  C.,  but  best  at  37°  C. 


SYMPTOMATIC  ANTHRAX. 


455 


The  artificial  cultivation  which  was  achieved  by 
Kitasato  is  not  more  difficult  than  that  of  other  an- 
aerobic organisms.  In  gelatin 
containing  i  to  2  per  cent,  of 
glucose  or  5  per  cent,  of  gly- 
cerin the  organism  develops 
quite  well,  the  exact  appearance 
depending  somewhat  upon  the 
method  by  which  it  was  planted. 
If  the  bacteria  are  dispersed 
through  the  culture -medium, 
the  little  colonies  will  appear 
in  the  lower  parts  of  the  tube  as 
nearly  spherical  or  slightly  irreg- 
ular, clouded,  liquefied  areas  con- 
taining bubbles  of  gas.  If,  on 
the  other  hand,  the  inoculation 
is  made  by  a  deep  puncture,  a 
stocking  -  shaped  liquefaction 
forms  along  the  whole  lower 
part  of  the  puncture,  leads  to 
considerable  gas-production,  and 
finally  causes  the  liquefaction  of 
all  the  gelatin  except  a  thin 
superficial  stratum, 
acid  odor  is  given  off  by  the 
cultures. 

When  the  bacteria  grow  anaerobically  in  Esmarch 
tubes,  the  colonies  are  irregularly  club-shaped  or  spheri- 
cal, with  a  tangled  mass  of  delicate  projecting  filaments 
visible  upon  microscopic  examination. 

In  agar-agar  the  development  is  similar  to  that  in 
gelatin.  The  gas-production  is  marked,  the  liquefaction 
of  course  absent,  and  the  same  acid  odor  pronounced. 

The  bacillus  also  develops  quite  well  in  bouillon,  the 
bacillary  masses  sinking  to  the  bottom  in  the  form  of 
whitish  flakes,  while  the  gas-bubbles  collect  at  the  top. 
In  this  medium  the  virulence  is  unfortunately  soon  lost. 


FIG.  129. — Bacillus  of  symp- 
A    nernliar   tomat^c  anthrax:  four-clays-old 
culture  in  glucose-gelatin  (Fran- 


456  PATHOGENIC  BACTERIA. 

Milk  also  seems  to  be  a  favorable  culture-medium. 
The  development  of  the  bacilli  is  unaccompanied  by 
coagulation. 

The  virulence  of  the  organism  is  soon  lost  in  all 
culture-media,  but  it  is  said  that  the  virulence  of  the 
culture  can  be  much  increased  by  the  addition  to  it  of 
20  per  cent,  of  lactic  acid. 

When  susceptible  animals  are  inoculated  with  a  minute 
portion  of  a  pure  culture  in  a  little  subcutaneous  pocket, 
such  as  is  described  in  connection  with  tetanus  and 
malignant  edema,  the  bacilli  proceed  to  grow,  pro- 
duce the  well-known  affection,  and  lead  to  a  certainly 
fatal  outcome.  Cows  seem  to  be  the  most  susceptible 
animals,  especially  those  between  six  months  and  four 
years  old ;  sheep  and  goats  are  also  sometimes  affected. 
Curiously  enough,  animals  that  are  immune  to  malig- 
nant edema  are  seemingly  more  susceptible  to  Rausch- 
brand.  Of  the  laboratory  animals,  the  guinea-pig  is 
most  susceptible ;  swine,  dogs,  and  rabbits  are  very 
slightly  susceptible ;  horses,  goats,  and  birds  are  im- 
mune. 

The  virulence  of  the  bacillus  is  capable  of  ready 
attenuation  by  exposure  to  heat,  by  previous  exposure 
of  its  spores  to  heat,  or  by  drying  combined  with  ex- 
posure to  increased  temperature.  The  inoculation  of 
animals  with  the  attenuated  bacilli  causes  a  very  mild 
affection,  followed  by  complete  immunity  to  the  viru- 
lent organisms.  Upon  this  principle  the  "  protective 
vaccination"  is  based.  Kitt  has,  however,  shown  that 
almost  the  same  method  as  that  employed  by  Pasteur 
for  vaccination  against  rabies  may  be  employed  against 
this  bacillus,  and  that  when  muscular  tissue  from  an 
animal  dead  of  the  disease  is  dried  at  a  temperature  of 
32~35°  C.,  and  then  exposed  for  six  hours  to  a  tempe- 
rature of  ioo°-io4°  C.,  and  a  second  portion  is  exposed 
in  the  same  manner  to  a  temperature  of  9O°-95°  C.,  an 
emulsion  of  this  tissue  in  distilled  water,  salt-solution, 
or  bouillon,  injected  into  the  animals  to  be  protected,  will 


SYMPTOMATIC  ANTHRAX.  457 

act  in  a  manner  resembling  the  pulverized  spinal  cords 
of  the  rabbits  used  in  rabies,  and  give  an  almost  per- 
fect immunity.  Roux  and  Chamberland  have  found  that 
filtered  cultures  can  also  produce  immunity  when  properly 
introduced  into  animals. 

The  immunity  to  symptomatic  anthrax  seems,  how- 
ever, to  be  one  of  degree,  for  Arloing,  Cornevin,  and 
Thomas  found  that  when  the  bacillus  was  introduced 
into  the  animal  body  simultaneously  with  a  20  per  cent, 
solution  of  lactic  acid,  either  the  virulence  of  the  bacil- 
lus or  the  resistance  of  the  tissues  was  so  changed  that 
natural  immunity  was  destroyed  and  the  bacteria  allowed 
to  develop  and  produce  the  disease.  Roger  found  also 
that  refractory  animals,  like  the  rabbit,  mouse,  pigeon, 
and  chicken,  could  be  made  susceptible  by  the  combined 
injection  of  the  Rauschbrand  bouillon,  the  Bacillus  pro- 
digiosus,  Proteus  vulgaris,  and  other  harmless  organisms. 

When  the  guinea-pig  is  inoculated  with  the  bacillus  of 
symptomatic  anthrax,  it  dies  in  from  twenty-four  to 
thirty-six  hours.  The  post-mortem  examination  shows 
a  bloody  serum  at  the  point  of  inoculation,  and  the  mus- 
cles are  dark  red  or  black,  like  those  of  the  ' '  black-leg ' ' 
of  cattle.  No  changes  are  apparent  in  the  internal  organs. 
The  bacilli  are  at  first  found  near  the  point  of  inocula- 
tion in  the  inflammatory  exudations  only,  but  soon  after 
death,  being  motile,  they  spread  to  all  parts  of  the  body. 

The  peculiarities  of  symptomatic  anthrax  point  to  the 
entrance  of  the  bacteria  into  the  animal  body  through 
wounds,  but  the  occurrence  of  epidemics  at  certain  geo- 
graphical points,  known  technically  as  "Rauschbrand 
stations,"  suggests  that  infection  may  also  take  place 
through  the  respiratory  and  alimentary  tracts. 

At  first  thought,  as  Frankel  points  out,  one  might 
imagine  that  an  animal  dead  of  quarter-evil  and  the  dis- 
charges from  its  body  might  be  harmless,  as  compared, 
for  example,  with  the  cadavers  and  discharges  of  anthrax, 
because  of  the  purely  anaerobic  method  of  the  growth  of 
the  bacillus  of  symptomatic  anthrax  and  the  rapidity  of  its 


458  PA  THOGENIC  BA  CTERIA. 

death  in  the  presence  of  oxygen.  This  is,  however,  un- 
true, for  the  rapid  development  of  a  permanent  form  in 
the  resisting  spores  of  the  bacillus  makes  the  pollution 
of  the  soil  exceedingly  dangerous  for  cows  who  subse- 
quently browse  upon  it.  That  the  spores  are  of  great 
vitality  is  shown  by  the  well-known  laboratory  method 
of  keeping  them  on  hand  for  experimental  purposes,  dried 
in  the  muscular  tissue  of  a  diseased  animal. 

Every  precaution  should  be  exerted  to  have  the  affected 
animals  isolated,  and  their  cadavers  disinfected  and  de- 
stroyed or  buried  in  such  a  manner  that  subsequent 
infection  is  impossible. 

Statistical  results  of  Guillod  and  Simon,  based  upon 
3500  protective  inoculations,  show  a  distinct  reduction 
of  the  death-rate  from  5-20  per  cent,  in  unprotected 
animals  to  0.5-2  per  cent,  in  protected  animals. 


CHAPTER    II. 
MALIGNANT   EDEMA. 

THE  chief  contaminating  organism  in  the  preparation 
of  pure  cultures  of  the  tetanus  bacillus  is  a  large  slender 
bacillus  almost  as  large  as  that  of  anthrax,  but  with 
rounded  ends  and  an  individual  motility  accomplished 
by  means  of  flagella  attached  to  its  ends  and  sides 
(Fig.  130).  It  is  a  strictly  anaerobic  bacterium,  and  was 


FIG.  130. — Bacillus  of  malignant  edema,  from  the  body -juice  of  a  guinea-pig 
inoculated  with  garden-earth;    x  1000  (Frankel  and  Pfeiffer). 

originally  described  by  Pasteur  (1875)  as  the  Vibrion 
septique.  It  grows  well  at  the  room-temperature,  as  well 
as  at  the  temperature  of  the  incubator,  produces  oval 
central  spores,  and,  because  of  its  association  with  a  spe- 
cific edema  in  certain  animals,  is  known  as  the  Bacillus 
cedema  maligni. 

459 


460  PA  THOGENIC  BA  CTERIA. 

The  organism  is  widely  distributed  in  nature,  being 
almost  always  present  in  garden-earth.  It  is  also  found 
in  various  dusts,  in  the  waste  water  from  houses,  and 
sometimes  in  the  intestinal  canals  of  animals. 

When  introduced  beneath  the  skin  this  bacillus  proves 
pathogenic  for  a  large  number  of  animals — mice,  guinea- 
pigs,  rabbits,  horses,  dogs,  sheep,  goats,  pigs,  calves, 
chickens,  and  pigeons.  Cattle  seem  to  be  immune. 

Giinther  points  out  that  the  simple  inoculation  of  the 
bacillus  upon  an  abraded  surface  is  insufficient  to  pro- 
duce the  disease,  because  the  oxygen  which  is,  of  course, 
abundant  there  is  detrimental  to  its  growth.  When  an 
experimental  inoculation  is  performed,  a  small  subcu- 
taneous pocket  should  be  made,  and  the  bacilli  introduced 
into  it  in  such  a  manner  as  not  to  be  in  contact  with  the 
air. 

If  the  inoculated  animal  be  a  mouse,  guinea-pig,  or 
rabbit,  in  about  forty-eight  hours  it  sickens  and  dies. 
The  autopsy  shows  a  general  subcutaneous  edema  con- 
taining immense  numbers  of  the  bacilli.  In  the  blood 
the  bacilli  are  few  or  cannot  be  found,  because  of  the 
oxygen  which  it  contains.  The  great  majority  of  them 
occupy  the  subcutaneous  tissue,  where  very  little  oxygen 
is  present  and  the  conditions  of  growth  are  therefore  good. 
If  the  animal  is  allowed  to  remain  undisturbed  for  some 
time  after  death,  the  bacilli  spread  to  the  circulatory  sys- 
tem and  reach  all  the  organs. 

Brieger  and  Ehrlich  have  reported  two  cases  of  malig- 
nant edema  in  man.  Both  cases  were  typhoid-fever 
patients  injected  with  musk,  and  developed  the  edema 
in  consequence  of  impurity  of  the  therapeutic  agent. 
No  case  is  reported,  however,  in  which  healthy  men 
have  been  infected  with  the  disease. 

Cornevin  declares  that  the  passage  of  the  bacillus 
through  the  white  rat  diminishes  its  virulence,  and  that 
the  animals  of  various  species  that  recover  from  this 
milder  affection  are  subsequently  immune  to  the  virulent 
organisms. 


MALIGNANT  EDEMA. 


461 


The  bacillus  of  malignant  edema  stains  well  with  ordi- 
nary cold  aqueous  solutions  of 
the    anilin    dyes,    but    not    by 
Grain's  method. 

The  organism  is  not  a  difficult 
one  to  secure  in  pure  culture, 
as  has  been  said,  generally  con- 
taminating tetanus  cultures  and 
being  much  more  easy  to  se- 
cure by  itself  than  its  congener. 
It  is  most  easily  obtained  from 
the  edematous  tissues  of  guinea- 
pigs  and  rabbits  inoculated  with 
garden-earth. 

The  colonies  which  develop 
upon  the  surface  of  gelatin  kept 
free  of  oxygen  appear  to  the 
naked  eye  as  small  shining 
bodies  with  liquid  grayish-white 
contents.  They  gradually  in- 
crease in  circumference,  but  do 
not  change  their  appearance. 
Under  the  microscope  they  ap- 
pear filled  with  a  tangled  mass 
of  long  filaments  which  under  a 
high  power  exhibit  individual 
movement.  The  edges  of  the 
colony  have  a  fringed  appearance,  much  like  the  hay  or 
potato  bacillus. 

In  gelatin  tube-cultures  the  characteristic  growth  can- 
not be  observed  in  a  puncture,  because  of  the  air  which 
remains  in  the  path  of  the  wire.  The  best  preparation 
is  made  by  heating  the  gelatin  to  expel  the  air  it  may 
contain,  inoculating  while  still  liquid,  then  replacing  the 
air  by  hydrogen,  and  sealing  the  tube.  In  such  a  tube 
the  bacilli  develop  near  the  bottom.  The  appearance  of 
the  growth  is  highly  typical,  as  globular  circumscribed 
areas  of  cloudy  liquefaction  result  (Fig.  131),  and  may  con- 


FlG.  131. — Bacillus  of  malig- 
nant edema  growing  in  glucose 
gelatin  (Frankel  and  Pfeiffer). 


462  PATHOGENIC  BACTERIA. 

tain  a  small  amount  of  gas.  In  gelatin  to  which  a  little 
grape-sugar  has  been  added  the  gas-production  is  marked. 
The  gas  is  partly  inflammable,  partly  CO2.  A  distinct 
odor  accompanies  the  gas-production,  and  is  especially 
noticeable  in  agar-agar  cultures. 


CHAPTER   III. 
BACILLUS   AEROGENES   CAPSULATUS. 

THIS  very  interesting  micro-organism  was  first  de- 
scribed by  Welch,  and  subsequently  carefully  studied  by 
Welch  and  Nuttall,1  and  Welch  and  Flexner.2  It  was 
first  secured  from  the  body  of  a  man  dying  suddenly  of 
aneurysm  with  a  peculiar  condition  of  gaseous  emphy- 
sema of  the  subcutaneous  tissue  and  internal  organs,  and 
a  copious  formation  of  gas  in  the  veins  and  arteries. 
The  blood  was  thin  and  watery,  of  a  lac-color,  and 
everywhere  contained  large  and  small  gas-bubbles.  The 
blood-alteration  was  associated  with  a  change  in  its 
coloring-matter,  which  dissolved  out  of  the  corpuscles 
and  stained  the  tissues  a  deep  red.  The  blood  was  found 
to  contain  many  bacilli,  which  were  also  obtained  from 
the  various  organs,  especially  in  the  neighborhood  of  the 
gas-bubbles.  The  bacilli  were  in  nearly  pure  culture. 

The  bacillus  is  a  large  organism,  measuring  3-5  //  in 
length,  about  the  thickness  of  the  anthrax  bacillus,  with 
ends  slightly  rounded,  or,  when  joined,  square  (Fig.  132). 
It  occurs  chiefly  in  pairs  and  in  irregular  masses,  but  not 
in  chains,  in  this  particular  differing  very  markedly  from 
the .  anthrax  bacillus.  In  culture-media  the  bacillus  is 
usually  straight,  with  slightly  rounded  ends.  In  old 
cultures  the  rods  may  be  slightly  bent,  and  involution- 
forms  occur.  When  several  bacilli  are  joined  together 
the  opposed  ends  are  square-cut.  The  bacillus  varies 
somewhat  in  size,  especially  in  length,  in  different  cul- 
ture-media. It  usually  appears  thicker  and  more  vari- 

1  Bull,   of  the  Johns  Hopkins  Hospital,  July  and  Aug.,   1892,  vol.   viii., 
No.  24. 

2  Jour,  of  Exper.  Med.,  vol.  i.,  No.  I,  Jan.,  1896. 

463 


464  PA  THOGENIC  BA  CTERIA. 

able  in  length  in  artificial  cultures  than  in  the  blood  of 
animals  and  of  man.  The  bacilli  occur  singly,  in  pairs, 
in  clumps,  and  sometimes  in  short  chains.  When  united, 
an  angle  is  often  formed. 

The  bacillus  is  non-motile  in  both  the  ordinary  hanging- 
drops  and  in  anaerobic  culture.  No  mention  is  made  of 
the  presence  of  flagella. 

Tlie  organism  stains  well  with  the  ordinary  stains,  and 
retains  the  color  well  in  Gram's  method.  When  stained 
with  methylene  blue  a  granular  or  vacuolated  appearance 


FIG.  132. — Bacillus  afirogenes  capsulatus  (from  photograph  by  Prof.  Simon 

Flexner). 

is  sometimes  observable,  due  to  the  presence  of  unstained 
dots  in  the  protoplasm. 

Usually  in  the  body-fluids  and  often  in  cultures  the  ba- 
cilli are  surrounded  by  distinct  capsules — clear,  unstained 
zones.  To  demonstrate  this  capsule  to  the  best  advan- 
tage, Welch  and  Nuttall  devised  the  following  special 
stain:  a  cover  is  thinly  spread  with  the  bacilli,  dried,  and 
fixed  without  over-heating.  Upon  the  surface  prepared, 
glacial  acetic  acid  is  dropped  for  a  few  moments,  then  al- 
lowed to  drain  off,  and  at  once  replaced  by  a  strong  aque- 
ous solution  of  gentian  violet,  which  is  poured  off  and 
renewed  several  times  until  the  acid  has  been  replaced  by 


BACILLUS  AEROGENES  CAPSULATUS.         465 

the  stain.  The  specimen  is  then  examined  in  the  color- 
ing-solution, after  soaking  up  the  excess  with  filter  paper, 
the  thin  layer  of  coloring  fluid  not  interfering  with  a  clear 
view  of  the  bacteria  and  their  capsules.  After  mounting 
in  Canada  balsam  the  capsules  are  not  nearly  so  distinct. 
The  width  of  the  capsule  varies  from  one-half  to  twice 
the  thickness  of  the  bacillus.  Its  outer  margin  is  stained, 
leaving  a  clear  zone  immediately  around  the  bacillus. 

It  was  at  first  thought  that  the  bacillus  produced  no 
spores,  but  Dunham1  found  that  spores  were  produced 
upon  blood-serum,  and  especially  upon  LofHer's  blood- 
serum  bouillon  mixture.  The  spores  resist  desiccation 
and  exposure  to  the  air  for  ten  months.  They  stain 
readily  in  hot  solutions  of  fnchsin  in  anilin  water,  and 
are  not  decolorized  by  a  moderate  exposure  to  the  action 
of  3  per  cent,  solution  of  hydrochloric  acid  in  absolute 
alcohol.  They  are  oval, -and  are  usually  situated  near 
the  middle  of  the  bacillus,  which  is  distended  because  of 
the  large  size  of  the  spore  and  bulges  at  the  sides. 

The  bacillus  is  anaerobic.  It  grows  upon  all  culture- 
media,  both  at  the  room-temperature  and  at  the  tempera- 
ture of  incubation,  best  at  the  latter.  The  bacillus  grows 
in  ordinary  neutral  or  alkaline  gelatin,  but  better  in  gela- 
tin containing  glucose,  in  which  the  characteristic  gas- 
production  is  marked.  Soft  gelatin,  made  with  5  instead 
of  10  per  cent,  of  the  crude  gelatin,  is  said  to  be  better 
than  the  ordinary  medium. 

There  is  no  distinct  liquefaction,  but  in  5  per  cent, 
gelatin  there  is  sometimes  a  softening  that  can  be  best 
demonstrated  by  tilting  the  tube  and  observing  that  the 
gas-bubbles  change  their  position,  as  well  as  by  noticing 
that  the  growth  tends  to  sediment. 

In  making  agar-agar  cultures  careful  anaerobic  precau- 
tions must  be  observed.  The  tubes  should  contain  con- 
siderable of  the  medium,  which  should  be  boiled  and 
freshly  solidified  before  using.  The  implantation  should 
be  deeply  made  with  a  long  wire.  The  growth  takes 

1  Bull,  of  the  Johns  Hopkins  Hospital,  April,  1897,  p.  68. 
30 


466 


PATHOGENIC  BACTERIA. 


.a, 


place  slowly  unless  such  tubes  are  placed  in  a  Buchner's 
jar.  The  deeper  colonies  are  the 
largest.  Sometimes  the  growth  only 
takes  place  within  10-12  mm.  of  the 
surface,  at  others  within  3-4  cm.  of 
it.  After  repeated  cultivation  the 
organism  seems  to  become  somewhat 
accustomed  to  the  presence  of  oxy- 
gen, and  will  grow  higher  up  in  the 
tube  than  when  freshly  secured  from 
animal  tissue  (see  Fig.  133). 

The  colonies  seen  in  the  culture- 
media  are  grayish-white  or  brownish- 
white  by  transmitted  light,  and  some- 
times exhibit  a  central  dark  dot.  At 
the  end  of  twenty-four  hours  the  larger 
colonies  do  not  exceed  0.5-1.0  mm. 
in  diameter,  though  they  may  subse- 
quently attain  a  diameter  of  2-3  mm. 
or  more.  Their  first  appearance  is 
as  little  spheres  or  ovals,  more  or  less 
flattened,  with  rather  irregular  con- 
tours, due  to  the  presence  of  small 
projecting  prongs,  which  are  quite 
distinct  under  a  lens.  The  colonies 
may  appear  as  little  irregular  masses 
with  projections. 

After  several  days  or  weeks,  single, 
well-separated  colonies  may  attain  a 
large  size  and  be  surrounded  by  pro- 
jections, either  in  the  form  of  little 
knobs  or  spikes  or  of  fine  branchings 
—hair-like  or  feathery.  Their  ap- 
pearance has  been  compared  to 
thistle-balls  or  powder-puffs  and  to 
thorn-apples.  When  the  growth 
takes  place  in  the  puncture  the 

feathery   projections   are    continuous.      Bubbles   of   gas 


FIG.  133.  —  Bacillus 
aerogenes  capsulutus, 
with  gas-production  (from 
photograph  by  Prof.  Si- 
mon Flexner). 


BACILLUS  AEROGENES  CAPSULATUS.        467 

make  their  appearance  in  plain  agar  as  well  as  in  sugar- 
agar,  though,  of  course,  less  plentifully.  They  first  ap- 
pear in  the  line  of  growth;  afterward  throughout  the 
agar,  often  at  a  distance  from  the  actual  growth.  Any 
fluid  collecting  about  the  bubbles  or  at  the  surface  of  the 
agar-agar  may  be  turbid  from  the  presence  of  bacilli. 
The  gas-production  is  more  abundant  at  incubation-  than 
at  room-temperatures. 

The  agar-agar  is  not  liquefied  by  the  growth  of  the 
bacillus,  but  is  often  broken  up  into  fragments  and  forced 
into  the  upper  part  of  the  tube  by  the  excessive  gas-pro- 
duction. 

In  its  growth  the  bacillus  produces  acid  in  considerable 
amount. 

In  bouillon  growth  does  not  occur  in  tubes  exposed  to 
the  air,  but  when  the  tubes  are  placed  in  Buchner's  jars, 
or  kept  under  anaerobic  conditions,  it  occurs  with  abun- 
dant gas-formation,  especially  in  glucose-bouillon,  with 
the  formation  of  a  frothy  layer  on  the  surface.  The 
growth  is  very  rapid  in  its  development,  the  bouillon 
becoming  clouded  in  two  to  three  hours.  After  a  few 
days  the  bacilli  sediment  and  the  bouillon  again  becomes 
clear.  The  reaction  of  the  bouillon  becomes  strongly 
acid. 

In  milk  the  growth  is  rapid  and  luxuriant  under 
anaerobic  conditions,  but  does  not  take  place  in  cul- 
tures exposed  to  the  air.  The  milk  is  coagulated  in 
from  twenty-four  to  forty-eight  hours,  the  coagulum 
being  either  uniform  or  firm,  retracted,  and  furrowed 
by  gas-bubbles.  When  litmus  has  been  added  to  the 
milk  it  becomes  decolorized  when  the  culture  is  kept 
without  oxygen,  but  turns  pink  when  it  is  exposed  to 
the  air. 

The  bacillus  will  also  grow  upon  potato  when  the  tubes 
are  enclosed  in  an  anaerobic  apparatus.  There  is  a 
copious  gas-development  in  the  fluid  at  the  bottom  and 
sides  of  the  tube,  so  that  the  potato  becomes  surrounded 
by  a  froth.  After  complete  absorption  of  the  oxygen  a 


468  PA  THOGENIC  BA  CTERIA . 

thin,  moist,  grayish-white  growth  takes  place  upon  the 
surface  of  the  potato. 

The  vital  resistance  of  the  organism  is  not  great.  Its 
thermal  death-point  was  found  to  be  58°  C:  after  ten 
minutes'  exposure.  Cultures  made  by  displacing  the  air 
with  hydrogen  are  less  vigorous  than  those  in  which  the 
oxygen  is  absorbed  from  the  air  by  pyrogallic  acid.  It 
was  found  that  in  the  former  class  of  cultures  the  bacillus 
generally  died  in  three  days,  while  in  the  absorption  ex- 
periments it  was  kept  alive  at  the  body-temperature  for 
one  hundred  and  twenty-three  days.  It  is  said  to  live 
longer  in  plain  than  in  sugar-agar.  To  keep  the  cultures 
alive  it  has  been  recommended  to  seal  the  agar-agar  tube 
after  two  or  three  days'  growth. 

It  is  believed  that  the  natural  habitat  of  the  bacterium 
is  the  soil,  but  there  is  reason  to  think  that  it  occurs  in 
the  intestine  at  times,  and  it  may  occasionally  be  found 
upon  the  skin. 

The  pathogenic  powers  of  the  bacillus  are  limited,  and 
while  in  some  cases  it  seems  to  be  the  cause  of  a  fatal 
outcome  in  infected  cases,  its  power  to  do  mischief  in  the 
body  seems  to  depend  upon  the  pre-existence  of  other 
depressing  and  devitalizing  conditions  predisposing  to  its 
growth. 

Being  anaerobic,  the  bacilli  are  unable  to  live  in  the 
circulating  blood,  but  they  grow  in  old  clots  and  in  cav- 
ities, such  as  the  uterus,  etc.,  where  but  little  oxygen 
ever  enters,  and  from  such  areas  enter  the  blood  and  are 
distributed. 

In  support  of  these  views  Welch  and  Nuttall  cite  the 
result  of  inoculation  into  healthy  and  diseased  rabbits. 
When  a  healthy  rabbit  is  injected  with  2l/2  c.cm.  of  a 
fresh  sugar-bouillon  into  the  ear-vein  it  generally  recov- 
ers without  any  evident  symptoms.  One  of  their  rabbits 
was  pregnant,  and  at  time  of  injection  was  carrying  two 
dead  embryos.  After  similar  injection  with  but  i  c.cm. 
of  the  culture  it  died  in  twenty-one  hours.  It  seems  that 
the  bacilli  were  first  able  to  secure  a  foothold  in  the  dead 


BACILLUS  AEROGENES  CAPSULATUS.        469 

embryos,  and  there  multiply  sufficiently  to  bring  about 
death  later  on. 

After  the  death  of  the  animal,  when  the  blood  is  no 
longer  oxygenated,  the  bacilli  grow  rapidly  with  a 
marked  gas-production,  which  in  some  cases  is  said  to 
have  caused  the  bodies  to  swell  to  twice  their  normal 
size.  The  result  of  injection  into  guinea-pigs  does  not 
differ  very  much  from  that  observed  in  rabbits.  Gaseous 
phlegmons  are  sometimes  produced. 

Pigeons  when  inoculated  subcutaneously  in  the  pec- 
toral region  frequently  succumb.  Following  the  injec- 
tion there  is  gas-production  that  causes  the  tissues  of  the 
chest  to  become  emphysematous.  The  bird  generally 
dies  in  from  seven  to  twenty-four  hours,  but  may  live. 

Intraperitoneal  inoculation  of  animals  sometimes 
causes  fatal  purulent  peritonitis. 

The  infection  as  seen  in  man  generally  occurs  from 
wounds  into  which  dirt  has  been  ground,  as  in  the  case 
of  a  compound,  comminuted  fracture  of  the  humerus, 
with  fatal  infection,  reported  by  Dunham,  or  in  wounds 
and  injuries  in  the  neighborhood  of  the  perineum. 

Among  the  twenty-three  cases  reported  by  Welch  and 
Flexner1  we  find  wounds  of  the  knee,  leg,  hip,  and  fore- 
arm, ulcer  of  the  stomach,  typhoid  ulcerations  of  the  in- 
testine, strangulated  hernia  with  operation,  gastric  and 
duodenal  ulcer,  perineal  section,  and  aneurism,  as  con- 
ditions in  which  external  or  gastro-intestinal  infection 
occurred. 

Dobbin,  P.  Ernst,  Graham  Stewart  and  Baldwin,  and 
Kronig  have  met  cases  of  puerperal  sepsis  and  sepsis  fol- 
lowing abortion  caused  by  the  bacillus,  or  in  which  it 
played  an  important  role. 

The  symptoms  following  infection  are  quite  uniform. 
There  are  usually  redness  and  swelling  of  the  wound, 
with  rapid  elevation  of  temperature  and  rapid  pulse.  The 
wound  is  usually  more  or  less  emphysematous,  and  dis- 
charges a  thin,  dirty,  brownish,  offensive  fluid  which  con- 

1  Jour,  of  Exper.  Meat.,  vol.  I,  No.  I,  Jan.,  1896. 


470  PA  THOGENIC  BACTERIA . 

tains  gas-bubbles  and  is  sometimes  frothy.  Occasionally 
the  patients  recover,  especially  when  the  infected  part  is 
susceptible  of  amputation,  but  death  is  a  more  common 
outcome.  After  death  the  body  begins  to  swell  almost 
immediately;  it  may  attain  twice  its  normal  size  and  be 
unrecognizable.  Upon  palpation  a  peculiar  crepitation 
can  be  felt  in  the  subcutaneous  tissue  nearly  everywhere, 
and  the  presence  of  gas  in  the  blood-vessels  is  easy  of 
demonstration.  The  gas  is  inflammable,  and  as  the  bub- 
bles ignite  explosive  sounds  are  heard. 

At  the  autopsy  the  gas-bubbles  are  found  in  most  of 
the  internal  organs,  sometimes  so  numerously  as  to  justify 
the  German  term  "  Schaumorgane "  (frothy-organs). 
The  liver  especially  is  apt  to  show  this  frothy  con- 
dition. When  the  tissues  from  such  a  case  are  hardened 
and  examined  microscopically  it  is  found  that  the  bub- 
bles appear  as  open  spaces  in  the  tissue,  the  "borders  of 
which  are  lined  with  large  numbers  of  the  gas  bacillus. 
There  are  also  clumps  of  bacilli  without  gas-bubbles,  but 
surrounded  by  tissue,  whose  nuclei  show  a  disposition  to 
fragment  or  disappear,  and  whose  cells  and  fibers  show 
signs  of  disintegration  and  fatty  change.  In  discussing 
these  changes  Ernst1  concluded  that  they  were  antc- 
mortem  and  due  to  the  irritation  caused  by  the  bacillus. 
The  gas-production  he  regarded  as  postmortem. 

In  the  internal  organs  the  bacillus  is  usually  found  in 
pure  culture,  but  in  the  wound  it  is  generally  mixed  with 
other  bacteria.  On  this  account  it  is  difficult  to  estimate 
just  how  much  of  the  damage  before  death  is  the  result 
of  the  activity  of  the  gas  bacillus.  That  gas-production 
after  death  has  nothing  to  do  with  pathogenesis  during 
life  is  shown  by  injecting  into  the  ear-vein  of  a  rabbit 
a  liquid  culture  of  the  gas  bacillus,  allowing  about  five 
minutes'  time  for  the  distribution  of  the  bacilli  through- 
out the  circulation,  and  then  killing  the  rabbit.  In  a  few 
hours  the  rabbit  will  swell  and  his  organs  and  tissues 
will  be  riddled  with  the  gas-bubbles. 

1  Virchow's  Archir,  U«I.  i.;;v  I  left  ii. 


BACILLUS  AEROGENES  CAPSULATUS.         471 

At  times,  however,  as  in  a  case  of  Graham  Stewart 
and  Baldwin,  there  is  no  doubt  that  the  bacillus  produces 
gas  in  the  tissues  of  the  entire  body  during  life.  These 
observers,  in  a  case  of  abortion  with  subsequent  infection, 
found  the  patient  "  enl^hysematous  from  the  top  of  her 
head  to  the  soles  of  her  feet"  several  hours  before 
death. 

In  this  case,  in  which  the  bacillus  was  found  in  pure 
culture,  it  would  indeed  be  difficult  to  doubt  that  the 
fatal  issue  was  due  to  the  bacillus  aerogenes  capsulatus. 
Whether  the  fatal  termination  of  the  cases  is  due  to  the 
presence  of  gas  in  the  vessels,  or  partly  to  that  and  partly 
to  some  toxic  property  it  possesses,  does  not  seem  to  have 
been  worked  out  as  yet.  It  would  seem,  however,  to  have 
a  toxic  property  from  the  fact  that  the  onset  of  the  infec- 
tion is  first  shown  by  the  occurrence  of  chill,  pyrexia, 
and  rapid  pulse,  and  from  the  change  caused  by  the 
clumps  of  bacilli  upon  the  surrounding  cells  of  the  tis- 
sues in  which  they  occur. 


CHAPTER    IV. 
BACILLUS  PROTEUS  VULGARIS  (HAUSER). 

THIS  bacillus  was  first  found  by  Hauser  in  decompos- 
ing animal  infusions,  generally  in  company  with  two 
closely  allied  forms,  Proteus  mirabilis  and  Proteus  Zen- 
keri,  which,  as  the  experiments  and  observations  of  San- 
felice  and  others  show,  may  be  identical  with  or  represent 


FIG.  134. 


-Swarming  islands  of  proteus  bacilli  on  the  surface  of  gelatin ;  x  650 
(Hauser). 


attenuated  forms  of  it.  According  to  Kruse,  it  is  quite 
probable  that  the  old  species  called  Bacterium  termo  was 
largely  made  up  of  the  proteus. 

The  bacilli  are  very  variable  in  size  and  shape — pleo- 
morphic— and  are  named  proteus  from  this  peculiarity. 
Some  forms  differ  very  little  from  cocci,  some  are  more 

472 


BACILLUS  PROTEUS    VULGARIS.  473 

like  the  colon  bacteria  in  shape,  others  are  found  as  very 
long  filaments,  and  occasionally  sporulina-forms  are  met 
with.  True  spirilla-forms  are  never  found.  All  the 
forms  mentioned  may  be  met  with  in  cultures  of  the 
same  organism.  "The  diameter  of  the  bacillus  is  usually 
about  0.6  py  but  the  length  varies  from  1.2  fj.  or  less  to  4  p 
or  more.  No  spores  are  formed.  The  organisms  are 
actively  motile.  The  long  filaments  frequently  form  loops 
and  tangles.  Flagella  are  present  usually  in  large  num- 
ber; upon  one  of  the  longer  bacilli  as  many  as  one  hun- 
dred have  been  counted.  Involution-forms  are  frequent 
in  old  cultures.  The  bacilli  stain  well  by  the  ordinary 
methods.  Gram's  method  is  irregular  in  action,  but 
usually  fails  to  color  the  bacteria. 

Upon  gelatin  plates  a  typical  phenomenon  is  observed 
in  connection  with  the  development  of  the  colonies,  but 
for  the  most  advantageous  observation  the  gelatin  used 
for  making  the  cultures  should  contain  only  5  per  cent, 
of  gelatin  instead  10  per  cent,  as  ordinarily  used.  Kruse L 
describes  the  phenomenon  as  follows:  "at  the  temperature 
of  the  room  rounded,  saucer-shaped  depressions,  with  a 
whitish  central  mass  surrounded  by  a  lighter  zone,  are 
quickly  formed.  Under  low  magnification  the  center  of 
the  growth  is  seen  to  be  surrounded  by  radiations  extend- 
ing in  all  directions  into  the  solid  gelatin,  and  made  up 
of  chains  of  bacilli.  Between  the  radiations  and  the 
granular  center  motile  bacteria  are  seen  in  active 
motion.  Upon  the  surface  the  colony  extends  as  a  thin 
patch,  consisting  of  a  layer  of  bacilli  arranged  in  threads, 
sending-  numerous  projections  from  the  periphery.  Occa- 
sionally filaments  are  found  in  the  surroundings.  Under 
certain  conditions  the  wandering  of  the  processes  can  be 
directly  observed  under  the  microscope.  It  depends  not 
only  upon  the  culture- medium,  but,  in  part,  upon  the 
culture  itself.  Entire  groups  of  bacilli  or  single  threads, 
by  gradual  extension  and  circular  movement,  detach 
themselves  from  the  colony  and  wander  about  upon  the 

1  Fliigge's  Microorganism  en. 


474  PA  THOGENIC  BA  CTERIA . 

plate.  Often  from  the  radiated  central  part  of  the  colony 
peculiar  zooglea  are  formed,  having  a  sausage-  or  screw- 
shape,  or  wound  in  spirals  like  a  corkscrew.  The 
younger  colonies,  which  have  not  yet  reached  the  surface 
of  the  gelatin,  are  more  compact,  rounded  or  nodular, 
later  covered  with  hair,  and  then  becoming  radiated  and 
like  the  superficial  colonies.'' 

When  the  culture-medium  is  more  concentrated,  or  the 
culture  one  that  has  been  frequently  transplanted,  the 
phenomenon  is  much  less  marked  and  sometimes  does 
not  take  place  at  all. 

Puncture-cultures  in  gelatin  are  not  at  all  character- 
istic. They  show  a  rapid  stocking-like  liquefaction  of 
the  gelatin,  extending  so  as  to  take  in  the  entire  gelatin 
in  the  tube  in  a  few  days.  Anaerobic  cultures  do  not 
liquefy. 

Upon  agar-agar  the  bacillus  grows  with  the  production 
of  a  moist,  thin,  transparent,  rapidly  extending  layer 
which  probably  rarely  reaches  the  sides  of  the  tube. 
Upon  agar-agar  plates  the  wandering  of  the  colonies  is 
also  said  to  occur. 

Upon  potato  the  growth  is  in  the  form  of  a  dirty-look- 
ing, smeary  patch. 

In  culture-media  containing  either  grape-  or  cane-sugar 
fermentation  occurs  both  in  the  presence  and  in  the 
absence  of  oxygen.  Milk-sugar  is  not  decomposed. 

When  grown  in  milk  the  medium  is  coagulated. 

In  its  growth  the  bacillus  usually  produces  a  strong 
alkaline  reaction.  Indol  and  phenol  are  formed  from 
the  peptone  of  the  culture-media.  Nitrates  are  reduced 
to  nitrites,  and  then  partly  reduced  to  NH3.  In  most 
culture-media  not  containing  sugar  the  bacillus  pro- 
duces a  very  disagreeable  odor. 

It  is  a  question  whether  the  Bacillus  proteus  is  to  be 
ranked  among  the  pathogenic  bacteria.  Small  doses  of 
it  are  harmless  for  the  laboratory  animals;  in  large  doses 
it  produces  abscesses.  A  toxic  substance  undoubtedly 
results  from  the  metabolism  of  the  organism,  and  is  the 


BACILLUS  PROTEUS    VULGARIS.  475 

cause  of  death  in  cases  in  which  considerable  quantities 
are  injected  into  the  peritoneal  cavity  or  blood-vessels. 
The  bacilli  do  not  seem  able  to  multiply  in  the  animal 
body  in  health,  but  can  do  so  when  there  has  been  pre- 
vious injury  to  its  tissues  or  when  associated  with  patho- 
genic bacteria.  In  such  cases,  if  it  be  enabled  to  grow 
in  considerable  quantity,  its  toxin  may  cause  pronounced 
symptoms.  By  various  observers  the  proteus  has  been 
secured  in  culture  from  cases  of  wound  and  puerperal  in- 
fections, purulent  peritonitis,  endometritis,  and  pleurisy. 
When  the  local  lesion  in  which  it  grows  is  small,  as  in 
endometritis,  the  danger  of  toxemia  is  slight,  but  when 
spread  over  large  areas,  as  the  peritoneum,  may  prove 
serious. 

It  is  quite  probable  that  in  some  of  the  cases  in  which 
blood-infection  with  the  proteus  has  been  found  after 
death  it  did  not  exist  previously,  as  the  researches  of 
Bordoni-Uffreduzzi  have  shown  that  the  proteus  quite 
regularly  enters  the  tissues  after  death. 

While  thus  apparently  unable  to  keep  up  an  indepen- 
dent existence  in  the  tissues  during  life,  and  important  in 
the  body  only  in  conjunction  with  other  bacteria,  the 
proteus  seems  able  to  grow  abundantly  in  urine  and  to 
produce  primary  inflammation  of  the  bladder  when  in- 
troduced spontaneously  or  experimentally  into  that  viscus. 
The  inflammatory  process  may  extend  from  the  bladder 
to  the  kidney,  and  so  prove  quite  serious. 

The  Bacillus  proteus  has  also  been  found  in  acute  in- 
fectious jaundice  and  in  acute  febrile  icterus,  or  Weil's 
disease. 


CHAPTER    V. 
WHOOPING-COUGH. 

IT  is  only  recently  that  the  bacteriology  of  whooping- 
cough  has  begun  to  assume  definiteness,  and  even  yet 
there  is  no  certainty  that  any  of  the  various  described 
bacteria  play  any  specific  part  in  its  etiology.  In  all 
diseases  of  the  respiratory  apparatus  the  discharges  are 
almost  certain  to  be  so  contaminated  with  the  nasal  and 
oral  bacteria  as  to  make  the  isolation  from  them  of  a 
single  probably  specific  organism  a  matter  of  difficulty, 
and  its  original  recognition  a  matter  of  genius. 

Of  historical  interest  are  the  researches  and  observa- 
tions of  Deichler,  KnrlofF,  Szemetzchenko,  Cohn,  Neu- 
mann, Ritter  and  Afanassiew.  Those  of  Kurloff  and 
Afanassiew  are  of  especial  importance  because  they  opened 
the  way  for  the  recent  studies  of  Koplik  l  and  those  of 
Czaplewski  and  Hensel.2  Koplik  and  Czaplewski  and 
Hensel  worked  entirely  independently  of  each  other,  and 
while  the  bacterium  studied  by  the  former  differs  in 
several  points  from  that  of  the  latter,  Czaplewski  and 
Hensel  have  claimed  to  see  in  Koplik's  work  a  confirma- 
tion of  their  own. 

Koplik  studied  16  cases  of  whooping-cough.  The 
sputum  was  collected  in  sterile  Petri  dishes,  in  which  it 
was  allowed  to  stand  for  an  hour  or  so  in  order  that  it 
should  break  up  into  mucous  fragments. 

When  the  clear  viscid  expectoration  from  uncompli- 
cated cases  of  whooping-cough  is  allowed  to  stand  for  an 

I  Centralbl.  f.  Bakt.   u.   Parasitenk.,  Sept.   15,  1897,  xxii.,  Nos.   8  and  9, 
p.  222. 

II  Deutsche  med.   Woch.,   1897,  No.   57,   p.   586,  and   Centralbl.  f.  Bakt.  u. 
Parasitenk.,  Dec.  22,  1897,  xxii.,  Nos.   22,  23,  p.  641. 


WHOOPING-COUGH.  .477 

hour  or  so  it  separates  into  a  fluid  portion  and  a  mass  of 
whitish,  opalescent,  irregularly  formed  flakes  or  frag- 
ments. These  were  selected  for  study,  and  were  trans- 
planted by  means  of  a  platinum-wire  hook  to  the  cul- 
ture-media. Czaplewski  and  Hensel  used  a  rather  better 
technique  than  this,  and  secured  purity  of  the  bacteria 
in  the  flakes  by  transferring  them  to  a  test-tube  contain- 
ing pepton  solution  and  violently  agitating  the  tube  to 
wash  off  foreign  bacteria.  After  washing,  the  flakes  were 
sown  upon  culture-media. 

Hydrocele-fluid  was  found  most  useful  as  a  culture- 
fluid,  but  particles  of  sputum  were  planted  upon  all 
the  culture-media,  and  attempts  to  cultivate  bacteria  from 
them  were  conducted  both  aerobically  and  anaerobically. 
In  13  out  of  the  16  cases  the  same  bacillus  (x)  was  iso- 
lated. The  organism  when  stained  and  examined  micro- 
scopically appeared  as  a  remarkably  short  and  delicate 
bacillus,  shorter  and  more  slender  than  the  diphtheria 
bacillus,  measuring  about  0.8-1.7  /J.  in  length  and  about 
0.3-0.4  ft  in  breadth.  When  stained  it  appeared  some- 
what granular,  and  so  resembled  somewhat  the  diphtheria 
bacillus.  Old  cultures  presented  similar  involution-forms 
to  those  seen  in  old  cultures  of  the  diphtheria  bacillus. 
In  general  the  bacillus  resembles  the  organism  found  by 
Afanassiew l  and  others  in  cover-glass  specimens  of 
whooping-cough  sputum,  but  differs  in  that  spores  were 
seen  several  times. 

In  pure  cultures  on  coagulated  hydrocele-fluid  the  ba- 
cillus forms  a  finely  granular  layer  of  pearl-white  color. 

On  agar-agar  the  cultures  are  opaque,  pearl-white,  and 
occur  as  a  thin  layer. 

The  colonies  iipon  agar-agar  are  whitish  by  reflected 
light,  and  straw-yellow  or  deeper  olive-green  by  trans- 
mitted light.  They  are  of  an  irregularly  rounded  shape 
and  are  granular. 

In  gelatin  puncture-cultures  the  growth  resembles  that 
of  the  streptococcus,  forming  along  the  track  of  the  wire 

1  St.  Petersbttrger  med.  Woch.,  1887,  Nos.  39-42. 


478  PA  THOGENIC  BA  CTERIA. 

a  line  of  finely  granular,  non-liquefying  colonies.  Upon 
the  surface  of  the  gelatin  the  growth  expands  so  as  to 
form  the  so-called  u  nail-growth." 

The  colonies  upon  gelatin  have  an  irregularly  circular 
form,  appear  white  or  straw-yellow  by  reflected  light  and 
olive-green  by  transmitted  light,  and  are  granular.  They 
do  not  liquefy  and  do  not  grow  to  large  colonies. 

In  bouillon  after  twenty-four  hours  there  was  a  faint 
clouding  of  the  liquid  and  subsequently  a  sedimentation 
of  the  bacteria  in  small  clusters.  After  a  week  or  so 
the  surface  of  the  medium  is  covered  with  a  delicate 
pellicle,  which  grows  thicker  with  the  passage  of  time. 

The  bacillus  grows  quite  well  anaerobically.  It  is 
motile. 

The  bacillus  is  pathogenic  for  mice,  but  does  not  pro- 
duce characteristic  symptoms  in  any  of  the  experiment- 
animals. 

In  discussing  the  results  of  Koplik's  work,  and  com- 
paring it  with  their  own,  which  very  shortly  preceded  it, 
Czaplewski  and  Hensel  suggest  that  the  bacillus  is  better 
described  as  a  bacterium  than  as  a  bacillus.  The  finely 
granular  ("fein  punktiertes  ")  appearance  described  by 
Koplik,  in  their  observations  seems  to  consist  of  a  deeper 
staining  at  the  poles  of  the  cells.  The  growths  on 
gelatin  and  on  Loffler's  blood-serum  mixture  correspond 
in  every  way.  The  agar-agar  growths  are  similar, 
though  a  slight  difference  in  color  is  noted,  and  is  attrib- 
uted to  a  difference  in  the  quality  of  the  medium  used. 
The  bouillon  culture  differs,  the  description  of  Czaplewski 
and  Hensel  being  as  follows:  at  the  end  of  a  day  at  37° 
C.  the  bouillon  is  scarcely  clouded.  At  the  bottom  of 
the  tube  is  a  sharply  defined,  lentil-like  sediment,  which 
arises  in  the  form  of  slimy  threads  when  the  fluid  is 
whirled  about,  and  mixes  with  the  fluid  when  ener- 
getically shaken.  Neither  bacillus  grows  on  potato. 
Koplik's  bacillus  was  also  peculiar  in  that  it  was  motile. 
Regarding  Koplik's  bacillus  as  identical  with  their  own, 
Czaplewski  and  Hensel  do  not  agree  with  him  in  believ- 


WHOOPING-COUGH.  479 

ing  it  to  be  the  same  as  that  described  by  Afanassiew, 
and  by  comparison  found  the  latter  to  be  a  much  larger, 
shorter,  more  elongate  bacillus.  Czaplewski  and  Hen- 
sel's  studies  embraced  44  cases  of  whooping-cough,  in 
which  the  bacillus  was  isolated  18  times;  5  cases  of 
bronchitis,  which  subsequently  developed  whooping- 
cough,  in  all  of  which  it  was  found;  and  i  case  of 
rhinitis  and  bronchitis  which  developed  whooping-cough, 
and  in  which  it  was  found  on  three  different  occasions. 

From  the  preceding,  it  will  be  seen  that  many  scholars 
have  labored  to  detect  the  specific  organism  of  this  dis- 
ease. At  present  several  agree  upon  the  presence  of«a 
certain  bacillus  in  the  expectorated  matter;  but  none  of 
them  have  yet  succeeded  in  producing  the  disease  or  any 
modification  of  it  in  the  lower  animals.  The  specificity 
is,  therefore,  a  matter  of  much  doubt,  and  rests  solely 
upon  the  constancy  of  the  presence  of  the  micro-organ- 
ism in  the  sputum. 


INDEX. 


ABBE  condenser  and  oil-immersion 
lenses,  hints  as  to  the  use  of, 
86 

Acid,  carbolic,  value  of,  as  a  germi- 
cide, 118 
Acids  and  alkalies,  production  of, 

by  bacteria,  54 
Actinomyces  bovis,  260 
Actinomycosis,  260 
fungus  of,  261 

growth  of,  262 
in  man,  263 
of  human  liver,  264 
resemblance  of,  to  tuberculosis, 

262 
Activity,  vital,  in  bacteria,  results 

of,  50 

Adhesion  preparation,  147 
Aerobic  bacteria,  45 
Aerogenic  bacteria,  50,  54 
Agar-agar    as    a    culture-medium, 

129 
advantages    of,    over  gelatin, 

150 

blood,  131 
hemoglobin,  131 
preparation  of,  129 
sedimentation  of,  130 
Air,  bacteriologic  examination  of, 

164 

Hesse's  apparatus,  165 
Petri's  filter  for,  166 
Sedgwick's  expanded  tube, 

167 

value  of,  167 
micro-organisms  in,  164 
pathogenic  bacteria  in,  164 
Alexin,  78 

Alkali  albuminate,  Deycke's,  133 
Alkaline  blood-serum,  133 
31 


Alkaloids,  animal,  51 

putrefactive,  51 
Anaerobic  bacteria,  45 
cultivation  of,  153 
cultures,  Novy's  jars  for,  156 
Anilin  dyes  and  bacteria,  affinity 

between,  90 
classification  of,  90 
employment   of,   in  study   of 

bacteria,  90 

for  bacteriological  work,  91 
introduction   of,   in    1877,    by 

Weigert,  26 
Animals,    experimentation    upon, 

158 
inoculation  of,  with  bacteria,  1 59, 

160 

to  secure  cultures,  148 
Anthrax,  356 
animals  most  frequently  affected 

by,  356 

antitoxin  of,  364 
bacillus  of,  357 

cultures  of,  359 

discovery  of,  356 

morphology  of,  357 

other  bacilli  resembling,  365 

pathogeny  of,  361 

resistant  powers  of,  363 

staining  of,  357 

susceptibility  of,  to  heat,  cold, 

etc.,  363 

foci  for  the  distribution  of,  356 
immunity  to,  experiments  in  de- 
struction of,  364 
in  cattle,  how  acquired,  364 

measures  to  prevent  the  spread 

of,  365 

means  by  which  infection   takes 
place,  361 

481 


482 


INDEX. 


Anthrax,  means  of  protecting  ani- 
mals against,  363,  364 
microscopic  examination  of  the 

various  organs  in,  362 
resistant  powers  of,  358 
spores,  357,  358,  360 
symptomatic,  453 
bacillus  of,  454 
cultures  of,  455 
staining  of,  454 
precautions  to  be  observed  in, 

458 
protective  inoculations  in,  456 

statistics  of,  458 
Antiabrin,  70 

Anti-infectious  substances,  8 1 
Antiphthisin,  236 
Antipneumococcic  serum,  351 
Antiricin,  79 
Antisepsis,  origin  of,  26 
Antiseptic    action,    results    of,    on 

bacteria,  178 
value  of  some  of  the  principal 

germicides,  113 
value  of  reagents,  determination 

of,  177 

Antistreptococcic  serum,  196 
Antitoxic  serum,  preparation  of,  for 

therapeutic  purposes,  302 
of  tetanus,  282 
Antitoxin  of  anthrax,  364 
of  cholera,  325 
of  diphtheria,  297 
of  tetanus,  282 
theory  of  immunity,  78 
Antitoxins,  79 
action  of,  upon  bacteria,  81 
origin  of,  80 

specific  for  one  disease  only,  83 
Antituberculin,  237 
Arnold's  steam  sterilizer,  108 
Aromatics,  production   of,  by  baC- 
trli.1,    56 

Arthrnsporcs,  35 

Ascococcus,  37 

Asiatic   cholera,  spirillum  of,   313, 

314 
Association,  effects  of,  on  bacteria, 

47 


Atmosphere    as    a    factor    in    the 
causation     of    suppuration, 

183 

bacteria  in,  183 
germs  in,  number  of,  167 
Autoclave,  1 1 1 
Trillat,  115 

BACILLI,  division  of,  38 
morphology  of,  38 
motility  of,  31 

Bacillus  aerogenes  capsulatus,  463 
colonies  of,  466 
cultures  of,  465 
gas- production  by,  467 
infection  of  man  by,  469 
morphology  of,  465 
natural  habitat  of,  468 
origin  of,  463 
pathogeny  of,  468 
staining  of,  464 
symptoms  produced  in  man 

by,  470 

vital  resistance  of,  468 
anthracis,  357 
colony  of,  146 
gelatin     puncture-culture     of, 

149 

spores  of,  358 

coli  communis,  200,  371,  389 
a  cause  of  cholera  intantuin, 

396 

cultures  of,  390,  391 
determination  of,  396 
differentiation  from  typhoid 

bacillus,  398 

immunization  against,  395 
in     drinking-water,     deter- 
mination of,  172 
in  yellow  fever,  399,  400 
pathogeny  of,  392-396 

for  animals,  394 
penetration      of      intestinal 

tissue,  392 
staining  of,  389 
varieties  of,  397 
coli  immobilis,  390 
colon,    389.     See    Bacillus    «>// 


INDEX. 


483 


Bacillus,  comma,  discovery  of,  29 
diphtheria.  284.  288,  289 

cover-glass     preparations     of, 

growth  of,  285.  286 
morphology  of,  284,  285 
staining 

toxin  elaborated  by,  297 
icteroides,  400 

antitoxic  serum  of,  408 

cultures  of,  402 

immunity  to,  408 

morphology  of,  401 

pathogenesis  of,  402 

specificity  in  man,  404 

staining  of.  402 

toxin  of,  404 
influenzas,  446 
Klebs-Loffler,  284 
lepro:.  242 

staining  of,  242 
liquefaciens    parvus.    colony   of. 

145 
mallei,  249 

cultivation  of,  250,  251 
staining  of,  . 

in  sections  of  tissue 
Kuhne's  method,  233 
Loffler's  method,  : 
mesentericus     vulgatus,    gelatin 

puncture-culture  of,  149 
mv.  lony  of,  146 

mycoides,  gelatin   puncture-cul- 
ture of,  149 

oedema  maligni.  459.  461 
of  Bordoni,  243 
of  bubonic  plague,  433 
of  chicken-cholera,  409 

of,  to  kill  rabbits,  412 
of  fowl-tuberculosi- 
growth  ot" 
staining  of.  239 
of  Friedlander,  352 
of  Havelburg,  405 
of  hog-cholera.  413 
of  Koplik,  47' 

of     malignant      edema,    gelatin 
puncture-culture  of".  14-) 


Bacillus  of  mouse-septicemia,  426, 

V* 

of  pseudo-diphtheria,  294 
of  pseudo-tuberculosis,  240 
of  rhinoscleroma,  273 
of  Sanarelli,  406 
of  swine-plague,  420,  421 
of  symptomatic  anthrax.  453 
inoculation  with,  456 
virulence  of,  456 
of  syphilis,  256 

Van  Niessen's,  257 
of  tetanus,  method  of  cultivating, 

277 

of  typhoid  fever,  370 
of  w  hooping-cough,  476,  477 
of  yellow  fever,  400 
pneumoniae,  352 
cultures  of,  353 
pathogen y  of,  354 
polypiformis,  colony  of,  145 
proteus  vulgaris,  472 

colonies  of,  472,  473 
cultures  of,  473,  474 
discover)-  of,  472 
found  in  human  body,  475 
morphology  of,  472 
pathogeny  of,  474 
pyocyaneus,  197 

crystal  formation  by,  199 

cultures  of,  198 

occurrence   in   human   being, 

199 

pathogeny  of,  199 
pyogenes  foetidus,  200 
radiatus,  colony  of,  145 
gelatin     puncture-culture    of, 

149 

suipestifer,  413 
suisepticus,  420 
tetani.  2-4 

colony  on  gelatin,  276 

cultures  of,  277 

distribution  of,  in  nature,  278 

isolation  of,  275 

means  of  entrance  into  animal 

•usm.  278 
puncture-culture  ot 
resistant  powers  of. 


484 


INDEX. 


Bacillus  tuberculosis,  blood-serum 
culture  of,  220 

channels  by  which  it  enters 
the  organism,  223 

chemotactic  property  of,  226 

difficulty  in  staining,  210,  211    \ 

infection  by,  222 

through  the  gastro-intestinal 

tract,  223 

through  the  placenta,  223 
through  the  respiratory  tract, 

223 

through    the    sexual    appa- 
ratus, 224 
through  wounds,  224 

isolation  of,  by  Koch,  204 

pure  cultures  of,  218 

relation  of  number  of,  in  spu- 
tum to  the  progress  of  the 
case,  214 

staining  of,  Ehrlich's  method, 

211 

Koch's  method,  211 
toxic  products  of,  229 
typhi,  366 

abdominalis,  370 
gelatin   puncture-culture  of, 

149 

and   bacillus    coli   communis, 
resemblance         between, 

371-375 

cultures  of,  369-375 
distribution  of,  in  nature,  368 
means   of    entrance   into   the 

body,  379 
morphology  of,  366 
murium,  423 
cultures  of,  423 
pathogenesis  of,  424,  425 
staining  of,  423 
resistant  powers  of,  369,  370 
staining  of,  367 

in  sections,  367 
typhoid,  means  of  entrance  into 

.  the  body,  379 

typhosus,  as  a  cause  of  suppura- 
tion, 200 
X,  400 

in  whooping-cough,  477 


Bacteria,  absence  of,  from   normal 
body-juices      and      tissues, 

43 

action  of  antitoxins  upon,  81 

aerobic,  45 

aerogenic,  50,  54 

anaerobic,  45 

cultivation  of,  153 
Botkin's  method,  156 
Buchner's  method,  153 
Esmarch's  method,  153 
Frankel' s  method,  1 54 
Gruber's  method,  154 
Hesse's  method,  153 
Liberius'  method,  153 
Ravenel's  method,  155 
Roux's  method,  157 
facultative,  45 
optional,  45 

and  anilin  dyes,  affinity  between, 
90 

and  spores,  difference  between, 

35 

biology  of,  43 

changes  in  cell-walls  of,  31 

changes  undergone  by,  in  process 
of  staining,  87 

chemical  analysis  of,  30 

chromogenesis  of,  52 

chromogenic,  52 

classification  of,  40,  50 
Cohn's  morphological,  42 

colonies    of,    appearance   under 

the  microscope,  145 
in    tubes,    Esmarch's    instru- 
ment for  counting,  171 

cover-glass  preparations  for  ex- 
amination of,  91 

cultivation  of,  124 

development  of,  in  liquids,  147 

distribution  of,  43 

elimination   of,  from   the  body, 

63 

entrance  of,  into  the  circulation, 
62 

examination  of,  in  solid  or  semi- 
solid  cultures,  89 

growing,  apparatus  for  examina- 
tion of,  89 


INDEX. 


485 


Bacteria,  growth  of,  conditions  in- 
fluencing, 45 

association  with  other  bac- 
teria., 47 
electricity,  47 
light,  46 
moisture,  46 
movement,  47 
nutriment,  45 
oxygen,  45 
reaction,  46 
temperature,  48 
.r-rays,  49 
in  gelatin,  148,  149 
in  air,  43,  183 

determination  of,  165 
number  of,  167 
quantitative    estimation    of, 

165 
in  body-juices  and  tissues  a  sign 

of  disease,  43 
influence  of  anilin  dyes  on,  30 

of  nuclear  stains  on,  30 
in  ice,  171 
injections  of,  into  animals,   158, 

159,  1 60 
in  milk,  57 
in  soil,  44,  174 

estimation  of  the   number  of, 

*75 
in    tissue,   Gram's    method    of 

staining,  97 
introduction  of,  into  animals,  by 

injection,   158-160 
in  water,  44 

apparatus    for    counting,    169, 

170 

filtration    as    a    means  of   di- 
minishing   the    number   of, 

172 
quantitative  determination  of, 

169,  170 
isolation  of,  139 
liquefaction  of  gelatin  by,  53 
locomotory  powers  of,  31 
measurement  of,  104 
methods  of  cultivating,  139 

Esmarch  tubes,  143 

Petri  dishes,  143 


Bacteria,    methods    of  cultivating, 
plate-cultures,   140 

of  observing,  86 
microscopic  examination  of,  145 
morphology  of,  36 
multiplication  of,  33 
non-chromogenic,  52 
non-pathogenic,  57 
of  specific  disease,  182 
organization  of,  40 
parasitic,  49 
pathogenic,  57 

in  the  air,  164 

means   of  entrance   into    the 

tissues,  58 

peptonization  of  mik  by,  56 
photogenic,  50,  56 
photographing  of,  104 
production  of  acids  and  alkalies 
by,  54 

of  aromatics  by,  56 

of  disease  by,  57 

of  gases  by,  54 

of  odors  by,  55 

of  phosphorescence  by,  56 
rate  of  development  of,  33 
recognition  of,  163 
reduction  of  nitrites  by,  56 
results  of  vital  activity  in,  50 
saprogenic,  50 
size  of,  32 

stained  or  unstained,   examina- 
tion of,  87 
staining  of,  in  sections  of  tissue, 

94 

Loffler's  method,  95 
Pfeiffer's  method,  96 
study  of,  in  the  stained  condition, 

90 

taken  in  respiration,  60 
that    do    not    stain    by    Gram's 

method,  99 

thermal  death-point  of,  176 
unstained,  method  of  examining, 

88 

weight  of,  33 
zymogenic,  50 
Bacteriologic   examination   of  air, 

value  of,  167 


486 


INDEX. 


Bacteriologic  examination  of  soil, 

174 

of  water,  169 
Bacteriology,  history  of,  17 
Bacterium,  38 

definition  of,  30 
Beef-peptone     in     preparation    of 

bouillon,  125 
Beggiatoa,  39 
Benches,  glass,  for  use  in  making 

plate-cultures,  142 
Binary  division,  33 

results  of,  33 

Birds,  susceptibility  of,  to  experi- 
mental     inoculation      with 
tubercle  bacilli,  239 
Black-leg,    453.       See    Anthrax, 

symptomatic. 
Black  vomit  of  yellow  fever,  cause 

of,  404 

Blood  agar-agar,  131 
Blood-serum,  alkaline,  133 
as  a  culture-medium,  131 
Koch's  apparatus  for  coagulating 

and  sterilizing,  133 
mixture,  Loffler's,  133 
therapy,  discovery  of,  29 
Body-juices,  antibactericidal  action 

of,  77 

Bordoni,  bacillus  of,  243 
Botkin's    apparatus    for      making 
anaerobic       plate  -  cultures, 
156 

Bottle    for      cultivating     tetanus- 
bacillus,  278 
Bouillon     as    a    culture-medium, 

124 

preparation  of,  124 
free  from  dextrose  as  a  culture- 
medium,  137 
Brown ian  movement,  32 
Bubonic  plague,  433 
antitoxin  of,  441 
bacillus  of,  435 
cultures  of,  435 
discovery  of,  29 
juthogenesis  of,  437,  438 
forms  of,  434 
serum,  441 


Buchner's  -method  of  making  an- 
aerobic cultures,  153 

CAPILLARY     TUBES    for    securing 
definite  quantities  of  blood 
in  typhoid  test,  381-390 
Carbol-fuchsin,  100 
Carbolic  acid,  value  of,  as  a  germi- 
cide, 118 

Carmin   and   hematoxylon,  efforts 
to   facilitate  observation  of 
bacteria  by  means  of,  90 
Catgut,  sterilization  of,  116 
Celloidin  as  an  imbedding  agent,  94 
Cells,  phagocytic,  71 
Charbon       symptomatique,      453. 
See     Anthrax,      symptom- 
atic. 

Cheese-poisoning,  cause  of,  51 
Chemotaxis,  71 
negative,  75 
Chicken-cholera,  409 
bacillus  of,  409 
cultures  of,  410 
pathogenesis  of,  411 
pneumococcus   of,   discovery  of, 

28 
Cholera,  311 

Asiatic,  spirillum  of,  312 
cultures  of,  315-318 
staining  of,  315 

effect  of  vaccination  in  preven- 
tion of,  325 

hog,  413 

immunity   to,    attempts    to   pro- 
duce, 324 
reasons  for,  320 
infectious  nature  of,  312 
spirillum   of,  in    drinking-water, 

means  of  detecting,  323 
pure   cultures    of,    Schottelius* 

method  of  securing,  316 
toxic   products  of  the  metab- 
olism of,  319 
theories  as  to  the  cause  of,  321, 

322 

Cilia,  31 

Circulation,  modes  of  entrance  of 
bacteria  into.  62 


INDEX. 


487 


Cladothrix,  39 

Closet,  hot-air,  107 

Clostridium,  34 

Clothing,  disinfection  of,  120 

Cocci,  36 

morphology  of,  36 
Cohn's   classification   of  the  bac- 
teria, 41,  42 
Comma     bacillus,     discovery    of, 

29 

Cotton,  sterile,  value  of,   in  bacte- 
riological work,  107 
Cover-glass  forceps,  93 

preparations  for   general   exam- 
ination, 91 
Gram's   method    for   staining, 

99 
method  of  fixing  material  for 

examination,  92 
Cover-glasses,  cleaning  of,  92 
Crystal-formation  by  bacillus  pyo- 

cyaneus,  199 

Cultivation  of  bacteria,  124 
Culture-media,  124 
agar-agar,   129 
alkaline  blood-serum,  133 
blood  agar-agar,  131 
blood-serum,  131 
bouillon,  124 
free  from  dextrose,  137 
Deycke's  alkali-albuminate,  133 
Eisner's,  371 
Dunham's  solution,  136 
gelatin,  127 
glycerin  agar-agar,  131 
hemoglobin  agar-agar,  131 
liquid,  best  means   of  keeping, 

127 
development   of   bacteria    in, 

H7 

litmus  milk,  135 
Lb'ffler's    blood-serum    mixture, 

133 

milk,  135 

peptone  solution,  136 
Petruschky's  whey,  136 
potato,  134 
potato-juice,  135 
sterilization  of,  108 


Cultures,  anaerobic,  various  meth- 
ods for  making,  153-157 
by  animal-inoculation,  148 
plate-,  140 
puncture-,  147 

in  gelatin,  various  appearances 

of,  149 
"pure,"  139 

method  of  making,  146 
solid  or  semi-solid,  examination 

of  bacteria  in,  89 
stroke-,  147 
study  of,  139 
Culture-tubes,    method    of   filling, 

126,   127 

method  of  inoculating,  141 
Cumol    for  sterilization  of  catgut, 

116 
Czenzynke's  staining-fluid,  447 

DAVAINE'S    classification    of     the 

bacteria,  41 

Death-point,  thermal,  176 
Dejecta,  sterilization  of,  119 
Deycke's  alkali-albuminate,  133 
Digestive  tract,  entrance   of  bac- 
teria into,  58 
Diphtheria,     antitoxic    serum     in 

treatment  of,  302-305 
determination     of    strength 

of,  303 

preparation  of,  302 
preservation  of,  302 
antitoxin,  297 

preparation  of,  298 
bacillus  of,  284,  288,  289 
discovery  of,  29 
relation  of,  to  diphtheria,  292 
susceptibility  of  different  ani- 
mals to,  293 
toxin  elaborated  by,  297 
bacteriologic  diagnosis  of,  287 
in   man   and    in    animals,    293, 

294 

pseudo-,  bacillus  of,  294 
Diplococci,  36,  37 
Diplococcus  of  mumps,  205 
cultures  of,  206 
morphology  of,  206 


INDEX. 


Diplococcus  pneumoniae,  346 

cultures  of,  347 

morphology  of,  346 

pathogenesis  of,  350 
Disease,  production  of,  by  bacteria, 

57 

Disease-germs,  isolation  and  culti- 
vation of,  28 

Diseases,  acute  inflammatory,  bac- 
teria of,  1 82 
chronic    inflammatory,    bacteria 

of,  208 

specific,  bacteria  of,  182 
toxic,  284 
Dishes,  Petri,  143 
Disinfection  of  clothing,  120 
of  furniture,  120 
of  patients,  122 

of  the  air  of  the  sick-room,  118 
of  the  skin,  115 

Disposal  of  the  bodies  of  persons 
dead  of  infectious  diseases, 
123 
Division,  binary,  of  bacteria,  33 

results  of,  33 
Drinking-water,  means  of  detecting 

cholera-spirilla  in,  323 
Dunham's   solution   as   a  culture- 
medium,  136 

Dyes,  anilin,  classification  of,  90 
introduction    of,   in    1877,   by 

Weigert,  26 
use  of,  in  study  of  bacteria,  90 

EDEMA,  malignant,  459 
bacillus  of,  459 

cultures  of,  461 

pathogenesis  of,  460 

staining  of,  461 
Ehrlich's  method  of  demonstrating 

the    presence     of    tubercle 

bacilli  in  sputum,  212 
of  staining  tubercle  bacilli  in 

sections  of  tissue,  216 
solution,  97 
Electricity,  influence  of,  on  growth 

of  bacteria,  47 
Elimination    of  bacteria  from  the 

body,  63 


Eisner's  culture-medium,  371 
method    of   separating   bacillus 

typhi  and  bacillus  coli  com- 

munis,  371 
Endocarditis,  ulcerative,  production 

of,  by  injection  of  staphylo- 
.  coccus  pyogenes  aureus,  189 
Endospores,  34 
Enzymes,  tryptic,  54 
Epidemic  parotitis,  204 
Erysipelas,  streptococcus  of,  194 
Esmarch  tubes,  143 
Esmarch's  instrument  for  counting 

colonies  of  bacteria  in  tubes, 

171 
method    of    making    anaerobic 

cultures,   153 
Examination,  bacteriologic,  of  air, 

164 

of  soil,  174 
of  water,  169,  170 
Excreta,  disinfection  of,  120 
Exhaustion    theory  of    immunity, 

70 
Experimentation     upon     animals, 

158 

FACTOKS  of  diphtheria  toxin,  300 
Farcin  du  bceuf,  270 

streptothrix  of,  270,  271 
Fermentation,   50 

-tube,  Smith's,  55 
Fetus,    infection    of,    through    the 
placenta,    by    the    bacillus 
tuberculosis,  223 

Fever,  relapsing,  431.     See  Relap- 
sing fever. 

splenic,  356.     See  Splenic  fever. 
typhoid,  366.  See  Typhoid  fever* 
yellow,  399.     See  Yellow  fever* 
Filter,  Kitasato's,  112 

Pasteur-Chamberland,  in 
Petri's,  for  air-examination,  166 
Reichel's,  1 12 
Filters,    porcelain,    sterilization  of, 

90 

Filtration  of  culture-media,  no 
various   substances    used   for, 
in 


INDEX. 


489 


Filtration  of  toxins,  apparatus  for, 

112 
Fiocca's  method  of  staining  spores, 

101 

Fission,  33 
Flagella,  31 
staining  of,  101 

conditions  essential  to  success 

in,  103 

Pitfield's  method,  103 
Forceps,  cover-glass,  93 
Formaldehyde  as  a  germicide,  114 
in  disinfection  of  rooms,  199,  122 
regenerator,  1 1 5 
Formalin,  114 

in  disinfection  of  rooms,  122 
use  of,  as  a  disinfectant,  115 
Fowl-tuberculosis,  bacillus  .of,  238 
Frankel's  instrument  for  obtaining 
earth   from   various    depths 
for   bacteriologic  study,  174 
method    of    making     anaerobic 

cultures,  154 
Friedlander's   method   of  staining 

bacteria  in  tissue,  97 
pneumonia  bacillus,  352 
cultures  of,  353 
pathogeny  of,  354 
Funnel  for  filling  tubes  with   cul- 
ture-media, 126 
Furniture,  disinfection  of,  121 

GABBETT'S  method  of  demonstrat- 
ing the  presence  of  tuber- 
cle bacilli  in  sputum,  214 

Gases,    production  of,  by  bacteria, 

54 

determination  of,  54 
Gelatin  as  a  culture-medium,  127 
growth  of  bacteria  in,  148,  149 
growth,    microtome    section  of, 

159,  1 60 

liquefaction  of,  by  bacteria,  53 
Generation,  spontaneous,   doctrine 

of,  18 

"Germ  theory  "  of  disease,  23 
Germicidal   value    of  gaseous  re- 
agents,    determination     of, 
1 80 


Germicidal  value  of  reagents,  deter- 
mination of,  178 
Koch's  method,  178 
Stern  berg's  method,  179 
Germicides,    chemical     action    of, 

117 

value  of  different,  113,  144 
Glanders,  248 
bacillus  of,  249 
cause  of,  248 

injections  of  mallein  in,  254 
Glassware,  sterilization  of,  106 
Glycerin    agar-agar    as   a   culture- 
medium,  131 

-gelatin   as   an    imbedding    me- 
dium, 85 

Gonococci,  cultivation  of,  202 
Gonococcus,  201 

in  urethral  pus,  201 
Gonorrhea,  201 

communication    of,    to    animals, 

203 
Gram's  method  of  staining  bacteria 

in  tissue,  97 

of  staining  cover-glass  prepa- 
rations, 99 
solution  for  staining  bacteria  in 

tissue,  98 

Gruber's  method  of  making  an- 
aerobic cultures,  154 

HANDS,  disinfection  of,  115 

Hanging-drop  method  of  examin- 
ing living  micro-organisms, 
88 

Havelburg's  bacillus,  405 

Heat,  moist,  in  sterilization  of  ap- 
paratus used  in  experi- 
mentation, 106 

use  of,  in  sterilization  of  instru- 
ments, etc.,  106 
|  Hemoglobin  agar-agar,  131 

Hesse's     apparatus   for   collecting 

bacteria  from  the  air,  165 
method    of    making    anaerobic 
cultures,  153 

Heyroth's  instrument  for  counting 
colonies  of  bacteria  in  Petri 
dishes,  170 


490 


INDEX. 


Hiss's  culture-media  for  differentia- 
tion    of     typhoid     bacillus 
from  allied  forms,  372 
History  of  bacteriology,  17 
Hog-cholera,  413 
bacillus  of,  413,  415 
culture  of,  415 
pathogen y  of,  417 
vitality  of,  417 
immunity  to,  418 
lesions  of,  414,  415,  417 
pathology  of,  414 
symptoms  of,  413 
Hot-air  closet,  107 
Humoral  theory  of  immunity,  75 
Hydrant-water,  number  of  bacteria 

in,  171 

Hydrophobia,  306 
and  tetanus,  parallelism  existing 

between,  307 
cure  for,  309 

incubation  period  of,  306 
treatment  of,    Pasteur's   system, 

309 

Hygienic  precautions  recommen- 
ded for  preventing  the  spread 
of  tuberculosis,  221,  222 

Hypodermic  syringes  for  introduc- 
tion of  bacteria  into  ani- 
mals, 158,  159 

ICE,  bacteria  in,  171 

-cream  poisoning,  cause  of,  51 
Imbedding  in  celloidin,  94 
in  glycerin-gelatin,  95 
in  paraffin.  95 
methods  of,  94 
Immunity,  acquired,  69 
and  susceptibility,  65,  66 
apparent,  67 

means  of  destroying,  67,  68 
natural,  66 
produced  by  antitoxins,   length 

of  83 

-reaction,  323 
theories  of,  70-83 
antitoxin,  78 
exhaustion,  70 
humoral,  75 


Immunity,    theories    of,   phagocy- 
tosis, 70 
retention,  70 

Immunizing  unit,  definition  of,  303 
Incubating  oven  for  use  in  cultiva- 
tion of  bacteria,  151 
Indol,  u,  56,  137,  319, 
Infection,    bacterial,    through    the 

digestive  tract,  58 
through  the  placenta,  62 
through  the  respiratory  tract, 

60 

through  the  skin  and  super- 
ficial mucous  membranes, 
61 

through  wounds,  62 
Influenza,  446 
bacillus  of,  446 

cultures  of,  447,  448 
discovery  of,  29 
pathogeny  of,  449 
staining  of,  447 
toxin,  effects   of,  when    injected 

into  animals,  450 
"Infusorial  life,"  21 
Injection  of  bacteria  into  animals, 

158,  159 
Injections  of  tuberculin,  results  of, 

231 
Instruments,  disinfection  of,  117 

sterilization  of,  106 
Intra-abdominal    and    intrapleural 
injections    for    introduction 
of    bacteria    into     animals, 
160 

Intravenous  injections  for  the  in- 
troduction of  bacteria  into 
animals,  159 

KASHIDA'S  method  of  differentiat- 
ing between  typhoid  ba- 
cillus and  bacillus  coli  com- 
munis,  372 

Kitasato's  filter,  1 1 1 

'•  Klatsch  priiparat,"  147 

Kny-Sprague  steam  sterilizer,  109 

Koch-Khrlich  method  of  demon- 
strating the  presence  of  tu- 
bercle bacilli  in  sputum,  2 12 


INDEX. 


491 


Koch's   apparatus  for  coagulating  I 
and  sterilizing  .blood-serum, 

133 
method  of  determining  the  germi- 

cidal  value  of  reagents,  178 
new  tuberculin,  231-236 

injection  of,   in  man,  234 
steam  apparatus  for  sterilization 

of  culture-medium,  108 
syringe,  159 

Koplick's  bacillus,  476-478 
Kiihne's     carbol-methylene    blue, 

253 

LENSES,  high-power,  use  of,  87 
low-power,  use  of,  87 
oil-immersion,  use  of,  87 
Leprosy,  241 
anesthetic,  247 
bacillus  of,  242 
cause  of,  241 

discovery  of,  28 
nodes  of,  246 
Leptothrix,  33,  39 
Leuconostoc,  38 
Levelling    apparatus    for    pouring 

plate-cultures,  140 
Liborius'  method  of  making  anaer- 
obic cultures,  153 
Life,    spontaneous    generation   of, 

doctrine  of,  18 

Ligatures,  disinfection  of,  116,  117 
Light,  influence  of,  on  growth  of 

bacteria,  46 

selection  of,  in  study  of  bacteria 
by  means  of  the  microscope, 
87 
Liquid  culture-media,  development 

of  bacteria  in,  147 
Liquids,  sterilization  of,  HI 
Listerism,  182 
origin  of,  27 
Litmus  milk  as  a  culture-medium, 

/35 
Loffler's  alkaline    methylene-blue, 

96 
blood-serum  mixture,  286 

as  a  culture-medium,  133 
method  of  staining  flagella,  101 


Loffier's  method  of  staining  sec- 
tions, 95 

Lugol's  solution,  dilute,  for  stain- 
ing bacteria  in  tissue,  97 

Lymphocytes,  71 

MADURA-FOOT,  266 
cause  of,  267 
streptothrix  of,  268 
Malignant  edema,  459 
Mallein,  254 

injections  of,  in  glanders,  254 
Measles,  451 
bacillus  of,  451 
cultures  of,  452 
discovery  of,  29 
staining  of,  451 
Meat-infusion,  125 
Meat-poisoning,  cause  of,  51 
Merismopedia,  36,  37 
Methods  of  observing  bacteria,  86 
Methyl-violet,  antiseptic   value  of, 
destructive    and  inhibitory, 
114 
Meyer's     bacteriological     syringe, 

'59 

Micrococci,  36 

Micrococcus  gonorrhceae,  201 
tetragenus,  200,  443 
cultures  of,  444 
pathogenesis  of,  445 
staining  of,  444 

Micro-organisms,  living,  hanging- 
drop  method  of  examina- 
tion, 88 

methods  of  destroying,  105 
on  the  skin,  183 
Microscope,   essential   features  of, 

86 
Microtome     sections      of     gelatin 

growths,  149 

Milk  as  a  culture-medium,  135 
as  a  medium  for  the  cultivation 
of  the  bacillus    diphtherias, 
291 

bacteria  in,  57 

peptonization  of,  by  bacteria,  56 
Mineral  salts,  effect  of,  in  bacterial 
cultures,  49 


492 


INDEX. 


Moisture,  influence  of,  on  growth 

of  bacteria,  46 
Mouse-holder,  161 
Mouse-septicemia,  426 
bacillus  of,  426 
cultures  of,  427 
pathogeny  of,  429 
staining  of,  429 
Movement,  Brownian,  32 

influence  of,  on  growth  of  bac- 
teria, 47 
Mumps,  204 

diplococcus  of,  205 
Mycetoma,  266 
streptothrix  of,  268 

cultures  of,  267 
Mycoderma,  148 
Myconostoc,  39 
Myco-phylaxin,  78 
Mycoprotein,  30 

composition  of,  30 
Myco-sozin,  78 

NASAL  mucous  membrane,  germi- 

cidal  power  of,  61 
Negative  chemotaxis,  75 
Nitrites,  reduction  of,  by  bacteria, 

56 
Novy's  jars  for  anaerobic  cultures, 

156 
Nutriment,  influence  of,  on  growth 

of  bacteria,  45 

ODORS,  production  of,  by  bacteria, 

55 

Ophidiomonas,  40 

Osteomyelitis,  production  of,  by  in- 
jection of  the  staphylococcus 
pyogenes  aureus,  189 

Oxygen,  influence  of,  on  growth 
of  bacteria,  45 

Oxy tuberculin,  236 

PARAFFIN  as    an   imbedding  me- 
dium, 95 
Parotitis,  epidemic,  204 

-in  (  hamberland  filter,  in 

treatment    of  hydropho- 
bia,  309 


Patients,  disinfection  of,  122 
Peptone  solution  as  a  culture-me- 
dium, 136 
Peptonization  of  milk  by  bacteria, 

Pest,  Siberian,  334.    See  Anthrax. 
Petri's  dishes,  143 

filter  for  air-examination,  166 
Petruschky's  whey,  136 
Pfeiffer's  method  of  staining  sec- 
tions, 96 
Phagocytes,  71 
Phagocytosis  theory  of  immunity, 

70 

Phenolphthalein   as  a  test  for  reac- 
tion of  culture-media,  125 
Phosphorescence,  production  of,  by 

bacteria,  56 

Photogenic  bacteria,  50,  56 
Phylaxins,  78 
Pigment-production,  52 
Pig  typhoid,  413 
Pitfield's   method   of  staining   fla- 

gella,   103 
Placenta,     entrance     of     bacteria 

through,  62 
Plague,  bubonic,  433.    See  Bubonic 

plague. 
Plate-cultures,  140 

anaerobic,  Botkin's  apparatus  for 

making,   156 

apparatus  for  making,  140 
drawbacks  to,  142 
method  of  making,  141 
Pneumobacillus,  352 
as  a  cause  of  suppuration,  200 
of  Frankel    and  Weichselbaum, 

200 
Pneumonia,  345 

bacillus  of,  352,  353 
catarrhal,  354 
lobar  or  croupous,  345 
diplococcus  of,  346 
cultures  of,  347 
morphology  of,  346 
pathogenesis  of,  350 
tubercular,  354 

Pneumonias,  complicating,  355 
mixed,  355 


INDEX. 


493 


Potato  as  a  culture-medium,  134 

-juice  as  a  culture-medium,  135 
Preparations,  cover-glass,  91,  92 

staining  of,  Gram's  method,  99 
Pseudodiphtheria,  bacillus  of,  294 
relation  of,  to  diphtheria,  295 
Pseudotuberculosis,  239 

bacillus  of,  240 
Ptomaines,  definition  of,  51 
Pump-water,    number  of   bacteria 

in,  171 
Puncture-cultures,  147 

gelatin,  various   appearance   of, 

149 

Pus,  urethral,  gonococcus  in,  201 
Putrefaction,  50 

QUARTER-EVIL,  453.  See  Anthrax, 
symptomatic. 

RABBITS,  method  of  making  intra- 
venous injections  into,  159 

Rabies,  306.     See  Hydrophobia. 

Rauschbrand,  453.     See  Anthrax, 
symptomatic. 

Ravenel's  method  of  making  an- 
aerobic cultures,  155 

Ray-fungus,  261 

Reaction,  influence  of,  on  growth 
of  bacteria,  46 

Reagents,    determination   of  anti- 
septic value  of,  177 
germicidal  value  of,  178 

Reichel's  filter,  1 1 1 

Relapsing  fever,  431 
spirillum  of,  431 

pathogenesis  of,  431,  432 
staining  of,  431 

Respiratory  tract,  entrance  of  bac- 
teria into,  60 

Results  of  vital  activity  in  bacteria, 

50 

chromogenesis,  52 
fermentation,  50 
liquefaction  of  gelatin,  53 
production    of  acids   and 
alkalies,  54 

of  aromatics,   56 

of  disease,  57 


'  Results  of  vital  activity  in  bacteria, 
production  of  gases,  54 
of  odors,  55 
of  phosphorescence,  56 
putrefaction,  50 
reduction  of  nitrites,  56 
Retention  theory  of  immunity,  70 
Rhinoscleroma,  2,73 

bacillus  of,  273 
River-water,  number  of  bacteria  in, 

171 

Roux's  bacteriological  syringe,  1 59 
method  of  cultivating  anaerobic 
bacteria,  157 

SALKOWSKI'S  method  of  determin- 
ing indol  in  cultures  of  ba- 
cillus coli,  392 

Salt  solution  as  a  disinfectant,  117 
Sanarelli's  bacillus,  400.     See  Ba- 
cillus icteroides. 
Saprogenic  bacteria,  50 
j  Saprophytes,  49 
I  Sarcina,  36,  37 
Schaumorgane,  470 
Schottelius'    method    of    securing 
pure  cultures   of  the   chol- 
era spirillum,  316 
Sedgwick's  expanded  tube  for  air- 
examination,  167 
Septic  diseases,  the,  431 
i  Serum,  anti-streptococcus,  176 
antitoxic,  of  anthrax,  364 
of  cholera,  325,  326 
of  diphtheria,  302 
of  tetanus,  282 

-test  for  typhoid  fever,  382,  383 
Siberian  pest,  453.     See  Anthrax. 
Sick-room,  disinfection  of,  118 
Skin  and  mucous  membranes,  en- 
trance of  bacteria  through, 
61 

disinfection  of,  115 
Smith's  fermentation-tube,  55 
Soil,  bacteria  of,  important,  175 

bacteriologic  examination  of,  174 
Solution,  Dunham's,  136 

Ehrlich's    anilin-water    gentian- 
violet,  212 


494 


INDEX. 


Solution,  Ehrlich's,  for  staining  bac- 
teria in  tissue,  97 
Solutions,  disinfecting,  uselessness 

of,  in  the  sick-room,  1 18 
staining-,  92 
Sozins,  78 
Spirilla,  39 

morphology  of,  39 

of  Philadelphia  waters,  344 

resembling  the  cholera  spirillum, 

326 

Spirillum  aquatilis,  341 
Berolinensis,  335 
Bonhoffi,  339 
cholera  Asiatica,  313-319 
characteristics  of,  319 
cultures  of,  315-318 
differentiation  of,  in  cultures, 

323,  324 

distribution  of,  319 
in  drinking-water,  means  of 

detecting,  323 
inoculation  forms  of,  314 
production  of  indol  by,  319 
resistant  powers  of,  322 
staining  of,  315 
toxic  products  of  the  metab- 
olism of,  319 
Danubicus,  337 
Denecke,  330 

cultures  of,  330 
Dunbar,  337 
Finkler  and  Prior,  326 
cultures  of,  327,  328 
staining  of,  329 
Gamaleia,  332 

cultures  of,  333,  334 
of  Gamaleia,    differentiation   of, 
from   spirillum   of    cholera, 

335 

pathogenesis  of,  335 
Metschnikofif,  332 
Milleri,  341 

of  Asiatic  cholera,  313-319 
trrrigenus,  342 
Weibeli,  340 

I.  of  \\Vrnirke,  338 

II.  of  \Vernicke,  338 
Spirorli.rta,  39 


Spirochaeta  febris  recurrentis,  431 

Spiromonas,  40 

Spirulina,  40 

Spleen,  influence  of,  on   the  vital 

resistance  to  disease,  68 
Splenic  fever,  356 
Spores,  34 

and  bacteria,  difference  between, 

35 
destruction    of,    by    intermittent 

sterilization,  109 
in  the  atmosphere,  183 
presence  of,  in  atmospheric  dust, 

105 

resistant  power  of,  35 
staining  of,  35,  100 

Fiocca's  method,  101 
Sporulation,  33.  34 

diagram  illustrating,  34 
Sputum,  tubercle  bacilli  in,  210 

demonstration  of,  210,  211 
!  Sputum-cup,  sanitary,  120 
;  Staining   bacteria   in    sections    of 

tissue,  94-97 
Loffler's  method,  95 
Pfeiffer's  method,  96 
cover-glass  preparations,  Gram's 

method  for,  99 
flagella,  method  of,  101 
Pitrleld's  method,  103 
fluid,  Czenzynke's,  447 
of  tubercle  tDacilli  in  sections  of 

tissue,  216 
solutions,  stock,  92 
spores,  i  oo 

Fiocca's  method,  101 
Staphylococci,  37 
Staphylococcus  epidermidis  albus, 

183 

41  golden,"  184 
pyogenes  albus,  184 

distribution    of,    in    nature, 

185 

growth  of,  1 86,  187 
staining  of,  186 
aureus,  185 
citreus,  189 

Steam,  sterilization  of  culture-media 
by,  1 08 


INDEX. 


495 


Steam  sterilizer,  Kny-Sprague,  109 
superheated,  for  quick   steriliza- 
tion of  culture-media,  1 10 
Sterilization  and  disinfection,  105 
fractional,  108 
intermittent,  109 
of  air  of  the  sick-room,  118 
of    blood-serum,    Koch's    appa- 
ratus for,   133 
of  culture-media,  108 
of  dejecta,  119 

of  instruments,  etc.,  used  in  ex- 
perimentation, 106 
of  liquids,  1 1 1 
of  porcelain  filters,  112 
of  surgical  dressings,   ligatures, 

etc.,  116,  117 
Sterilizer,  Arnold's,  108 
hot-air,  107 
Kny-Sprague,  109 
Koch's,  108 
Sternberg's  milk,  177 

method  of  determining  germi- 
cidal  value  of  reagents, 
179 

Stock-solutions  for  staining,  92 
Streptococci,  36,  37 

in    intestinal    canal    of   infants, 

'93 

Streptococcus  conglomeratus,  191 
diffusus,  191 
erysipelatis,  194 

as  a  therapeutic    measure   in 

treatment  of  tumors,  196 
longus,  190 
pyogenes,  190 
growth  of,  191 
staining  of,  190 
virulence  of,  192 
vitality  in  culture,  191 
Strepto-diplococcus,  37 
Streptothrix,  39 
Madurse,  268 

cultures  of,  267,  268 
of  farcin  du  bceuf,  270,  271 
Stroke-cultures,  147 
Subcutaneous  injections  for  the  in- 
troduction  of  bacteria   into 
animals,  158 


Sucholo-albumin,  418 
Sucholotoxin,  418 
Suppuration,  182 

air  as  a  factor  in   the   causation 

of,  183 

Suppuration,  causes  of,  183 
Surgery,  antiseptic,  182 
Sutures,  disinfection  of,  117 
Swine-plague,  420 
bacillus  of,  420,  421 
culture  of,  422 
pathogenesis  of,  422 
staining  of,  422 
lesions  in,  421 
symptoms  of,  421 
Symptomatic  anthrax,  453 
Syphilis,  255 
bacillus  of,  256 

staining  of,  255,  256 
Van  Niessen's,  257 
Syringes,  disinfection  of,  159 
for   subcutaneous    injections    of 
bacteria  into  animals,    158, 
'59 

TEMPERATURE,    influence    of,    on 

growth  of  bacteria,  48 
Tetanin,  280 
Tetano-toxin,  280 
Tetanus,  274 

and     hydrophobia,     parallelism 

existing  between,  307 
antitoxic    serum    of,   preparation 

of,  282 

therapeutic  value  of,  282 
bacillus  of,  274 
cultures  of,  277 
discovery  of,  29 
distribution  of,  in  nature,  278 
method  of  cultivating,  277 
-bottle,  278 
pathology  of,  281 
susceptibility  to,  of  different  ani- 
mals, 279 

-toxin,  nature  of,  280 
preparation  of,  281 
Tetragenococci,  36,  37,  443 
Thermal    death-point  of  bacteria, 
determination  of,  176 


496 


INDEX. 


Toxin   elaborated   by  the  bacillus 

diphtherias,  297 

Toxins,  rapid   filtration   of,   appa- 
ratus for,  1 1 2 
Toxo-phylaxin,  78 
Toxo-sozin,  78 
Trillat  autoclave,  1 1  5 
TR-tuberculin,  233 

injection  of,  in  man,  234 
objection  to,  235 
Tubercle  bacilli,  209 

channels  by  which  they  enter 

the  organism,  223 
cultivation  of,  217-221 
discovery  of,  28 
growth  of,  219 
in  sections  of  tissue,  216,  217 
methods  of  demonstrating 
the    presence    of,    216, 
217 
in   sputum,  demonstration  of, 

211,  212 

Ehrlich's  method,  212 
Gabbett's  method,  214 
Koch-Ehrlich  method,  2 1 2 
Ziehl's  method,  213 
pure  cultures  of,  218 
toxic  products  of,  226,  229 
Tubercles,  226,  227,  228 
Tuberculin,  230 
action  of,  230 
Koch's  new,  231-236 
preparation  of,  231 
result  of  the  injection  of,  231 
TO,  233 
TR,  233 

Tuberculosis,  208 
bacillus  of,  209-229.   See  Tuber- 
cle bacilli. 
discovery  of,  28 
fowl-,  238 
gallinarum,  238 

hygienic      precautions      recom- 
mended for  preventing  the 
spread  of,  222,  223 
latent,  229 

macroscopic  lesions  of,  224 
Tuberculous      patients,       sanitary 
sputum-cup  for  use  of,  120 


i  Tube,  Sedgwick's,  for  air-examina- 
tion, 167 
Tubes,  Esmarch,  143 

for   securing    definite  "quantities 
of   blood    for    typhoid   test, 

384-390 

Tumors,  treatment  of,  by  inocula- 
tion  with  the  streptococcus 
erysipelatis,  196 
Tyndall  on  the  "  germ  theory  "  of 

disease,  25 
Typhoid  fever,  366 
bacillus  of,  366 
cultures  of,  369 
differentiation  of,  from  bacil- 
lus  coli   communis,  371- 
379-  402 
discovery  of,  28 
resistant  powers  of,  368 
staining  of,  367 
comparative  immunity  of  ani- 
mals to,  379-382 
inoculation      experiments    on 

animals,  379 
prophylaxis  in,  379 
Widal's    serum-test    for,    386, 

387 

Pig-,  413 

serum,  action  of,  381 
Typhotoxin,  378 
Tyrotoxicon,  51 

UNNA'S  method  of  staining  tubercle 
bacilli  in  sections  of  tissue, 
216 

VAN    NIESSEN'S    syphilis-bacillus, 

257 
Vibrio,  39 

Schuylkiliensis,  342 
colonies  of,  342 
growth  of,  343 
pathogeny  of,  343 
Vital  activity  in  bacteria,  results  of, 
50 

WATKR,    bacteria   in,    quantitative 

determination  of,  169 
bacteriologic  examination  of,  169 


INDEX. 


497 


Whey,  Petruschky's,  136 
Whooping-cough,  476 
bacillus  of,  476,  477 
bacillus  Xin,  477 
Widal  serum-test  for  typhoid  fever, 

382,  383 
Wolfhiigel's  apparatus  for  count- 

ing colonies  of  bacteria  upon 

plates,  169 
Wooden  tongue,  265 
Wounds,  unprotected,  entrance  of 

bacteria  into,  62 


,    effects  of,  on  growth  of 
bacteria,  49 

YEAST-PLANT  as  the  cause  of  fer- 
mentation, discovery  of,  27 
32 


Yellow  fever,  399 

antitoxic  serum  of,  408 
bacillus  of,  400.     See  Bacillus 

icteroides. 

coli  communis  in,  399,  400 
bacillus  X  in,  400 
cause  of  black  vomit  in,  404 
causes  of  death  in,  403 
Havelburg's  bacillus  in,  405 
pathology  of,  405 

ZIEHL'S  method  of  demonstrating 
the  presence  of  tubercle  ba- 
cilli in  sputum,  213 

Zooglea,  148 

Zoph's  classification  of  the  bacteria, 

4i 
Zymogenic  bacteria,  50 


CATALOGUE 

OF  THE 

MEDICAL  PUBLICATIONS 


OF 


.  B.  SAUNDERS, 

No.   925   WALNUT   STREET,   PHILADELPHIA. 
Arranged  Alphabetically  and  Classified  under  Subjects. 


THE  books  advertised  in  this  Catalogue  as  being  sold  by  subscription  are  usually  to  be 
obtained  from  travelling  solicitors,  but  they  will  be  sent  direct  from  the  office  of  pub- 
lication (charges  of  shipment  prepaid)  upon  receipt  of  the  prices  given.     All  the  other 
books  advertised  are  commonly  for  sale  by  booksellers  in  all  parts  of  the*  United  States;  but 
books  will  be  sent  to  any  address,  carriage  prepaid,  on  receipt  of  the  published  price. 

Money  may  be  sent  at  the  risk  of  the  publisher  in  either  of  the  following  ways :  A  post- 
office  money  order,  an  express  money  order,  a  bank  check,  and  in  a  registered  letter.  Money 
sent  in  any  other  way  is  at  the  risk  of  the  sender. 

See  pages  30,  3  J,  for  a  List  of  Contents  classified  according  to  subjects. 

— . — 

LATEST  PUBLICATIONS. 


American  Text-Book  of  Dis.  of  Eye,  Ear,  Nose,  and  Throat.    Page  3. 

American  Text-Book  of  Genito-Urinary  and  Skin  Diseases.    Page  4. 

American  Text-Book  of  Diseases  of  Children — Rev.  Edition.    Page  3. 

American  Text-Book  of  Gynecology — Revised  Edition.     See  page  4. 

American  Year-Book  of  Medicine  and  Surgery.     See  page  6. 

Anders'  Practice  of  Medicine — Revised  Edition.     See  page  6. 

Vierordt's  Medical  Diagnosis — Fourth  (Revised)  Edition.    See  page  29. 

Kyle  on  the  Nose  and  Throat.     See  page  15. 

Church  and  Peterson's  Nervous  and  Mental  Diseases.     See  page  8. 

Da  Costa's  Surgery — Revised  and  Enlarged  Edition.     See  page  JO. 

Saunders'  Medical  Hand-Atlases.     See  page  2. 

Griffith  on  The  Baby — Revised  Edition.     See  page  12. 

Butler's  Materia  Medica  and  Therapeutics — Revised  Edition.     Page  8. 

De  Schweinitz'  Diseases  of  the  Eye— Revised  Edition.     See  page  10. 

Vecki's  Sexual  Impotence.    See  page  28. 

Stoney's  Materia  Medica  for  Nurses.     See  page  28. 

Penrose's  Diseases  of  Women — Second  Edition.     See  page  18. 

McFarland's  Pathogenic  Bacteria — Revised  Edition.    See  page  17 

American  Pocket  Medical  Dictionary.    See  page  10. 

Stengel's  Text-Book  of  Pathology.    See  page  26. 

Hirst's  Text-Book  of  Obstetrics.     See  page  13. 

Grafstrom's  Massage  and  Medical  Gymnastics.    Page  12. 

Saunders'  Pocket  Formulary— Fifth  (Revised)  Edition.     See  page  24. 

Stevens'  Practice  of  Medicine— Fifth  (Revised)  Edition.     See  page  27. 


SAUNDERS'  MEDICAL  HAND-ATLASES. 


THE  series  of  books  included  under  this  title  consists  of  authorized  translations  into 
English  of  the  world-famous  Lehmann  Medicinische  Handatlanten,  which  for  sci- 
entifc  accuracy,  pictorial  beauty,  compactness,  and  cheapness  surpass  any  similar 
volumes  ever  published.  Each  volume  contains  from  50  to  100  colored  plates,  executed 
by  the  most  skilful  German  lithographers,  besides  numerous  illustrations  in  the  text.  There 
is  a  full  and  appropriate  description  of  each  plate,  and  each  book  contains  a  condensed 
but  adequate  outline  of  the  subject  to  which  it  is  devoted. 

One  of  the  most  valuable  features  of  these  atlases  is  that  they  offer  a  ready  and  satis- 
factory substitute  for  clinical  observation.  To  those  unable  to  attend  important  clinics 
these  books  will  be  absolutely  indispensable. 

In  planning  this  series  ot  books  arrangements  were  made  with  representative  publishers 
in  the  chief  medical  centers  of  the  world  for  the  publication  of  translations  of  the  atlases 
into  nine  different  languages,  the  lithographic  plates  for  ail  these  editions  being  made  in  Ger- 
many, where  work  of  this  kind  has  been  brought  to  the  greatest  perfection.  The  expense  of 
making  the  plates  being  shared  by  the  various  publishers,  the  cost  to  each  one  was  materially 
reduced.  Thus  by*reason  of  their  universal  translation  and  reproduction,  the  publish- 
ers have  been  enabled  to  secure  for  these  atlases  the  best  artistic  and  professional 
talent,  to  produce  them  in  the  most  elegant  style,  and  yet  to  offer  them  at  a  price 
heretofore  unapproached  in  cheapness.  The  success  of  the  undertaking  js  demon- 
strated by  the  fact  that  the  volumes  have  already  appeared  in  nine  different  languages 
— German,  English,  French,  Italian,  Russian,  Spanish,  Danish,  Swedish,  and  Hungarian. 

In  view  of  the  striking  success  of  these  works,  Mr.  Saunders  has  contracted  with  the 
publisher  of  the  original  German  edition  for  one  hundred  thousand  copies  of  the  atlases. 
In  consideration  of  this  enormous  undertaking,  the  publisher  has  been  enabled  to  prepare 
and  furnish  special  additional  colored  plates,  making  the  series  even  handsomer  and  more 
complete  than  was  originally  intended. 

As  an  indication  of  the  practical  value  of  the  atlases  and  of  the  favor  with  which  they 
have  been  received,  it  should  be  noted  that  the  Medical  Department  of  the  U.  S.  Army 
has  adopted  the  "Atlas  of  Operative  Surgery  "  as  its  standard,  and  has  ordered  the  book  in 
large  quantities  for  distribution  to  the  various  regiments  and  army  posts. 

The  same  careful  and  competent  editorial  supervision  has  been  secured  in  the 
English  edition  as  in  the  originals,  the  translations  being  edited  by  the  leading  American 
specialists  in  the  different  subjects. 

NOW  READY. 

Atlas  of  Internal  Medicine  and  Clinical  Diagnosis.  By  DR.  CHR.  JAKOB,  of  Erlangen.  Edited 
by  AUGUSTUS  A.  ESHNER,  M.D.,  Professor  of  Clinical  Medicine  in  the  Philadelphia  Policlinic;  At- 
tending Physician  to  the  Philadelphia  Hospital.  68  colored  plates,  and  64  illustrations  in  the  text. 
Cloth,  13.00  net. 

Atlas  of  Legal  Medicine.  By  DR.  E.  R.  VON  HOFMANN,  of  Vienna.  Edited  by  FREDERICK  PETER- 
SON, M.D..  Qlinical  Professor  of  Mental  Diseases,  Woman's  Medical  College,  New  Vork  ;  Chief 
of  Clinic,  Nervous  Dept.,  College  of  Physicians  and  Surgeons,  New  York.  With  120  colored  fig- 
ures on  56  plates,  and  193  beautiful  halt-tone  illustrations.  Cloth,  $3.50  net. 

Atlas  of  Diseases  of  the  Larynx.     By   DR.   L.  GRUNWALD,  of  Munich.    Edited  by  CHAKI 

GRAYSON,  M.D.,  Lecturer  on  Laryngology  and  Khinology  in  the  University  of  Pennsylvania; 
Physician-in-Charge,  Throat  and  Nose  Department,  Hospital  of  the  University  of  Pennsylvania* 
With  107  colored  figures  on  44  plates,  and  25  text-illustrations.  Cloth,  12.50  net. 

Atlas  of  Operative  Surgery.  By  DR.  O.  ZUCKFRKANDL,  of  Vienna.  Edited  by  J.  CHALMKRS 
DACOSTA,  M.D.,  Clinical  Professor  of  Surgery.  Je he i son  Medical  College,  Philadelphia ;  Sni.^<>" 
to  the  Philadelphia  Hospital.  With  24  colored  plates,  and  217  text  illustrations.  Cloth,  $3.00  net. 

Atlas  of  Syphilis  and  the  Venereal  Diseases.  By  PROF.  DR.  FRANZ  MRACHK,  of  Vienna.  Edited 
by  L.  HIM  i»N  HANGS,  M.  D.,  Professor  of  Genito-Urinary  Surgery,  University  and  Bellevue  Hospi- 
tal Mi-dic.d  Coll.--.  .  V«  \  irk.  With  71  colored  plates,  n>  black-and-white  illustrations,  and  12* 
pages  of  text.  Cloth,  £3 

Atlas    of    External    Diseases    of    the    Eye.       I'.y    Di<     O.    HAAB,  of   Zurich.       Edited    by    < 

in  >I-II\VKINIT/,  M.  !>.,  Professor  of  Ophthalmology,  _l'-tt<-i>  n  Medical  College.  Philadelphia. 
With  y'l  colon-d  illustrations  on  40  pl.ite^,  and  228  pages  of  text.  Cloth,  £3.00  net. 

Atlas  of  Skin  Diseases.  I'.y  Pi:  IF.  DK.  KKANZ  MRACEK,  of  Vienna.  Kdiicd  l.y  HI-NKY  W.  STI-L WAGON, 
M  1»  .Clinical  l'i  rmatology,  Jefienon  Medical  College,  Philadelphia.  63  colon.. 

39  be.iutiful  half-one  ilhisti  .iti"ti>,  and  200  pages  of  text.     CL>th,  #3.50  net. 

IN  PREPARATION. 

Atlas  of  Pathological  Histology.  Atlas  of  Operative  Gynecology. 

Atlas  of  Orthopedic  Surgery.  Atlas  of  Psychiatry. 

Atlas  of  General  Surgery.  Atlas  of  Diseases  of  the  Ear. 


THE  AMERICAN  TEXT-BOOK  SERIES. 
AN  AMERICAN  TEXT-BOOK  OF  APPLIED  THERAPEUTICS. 

By  43  Distinguished  Practitioners  and  Teachers.  Edited  by  JAMES  C. 
WILSON,  M.D.,  Professor  of  the  Practice  of  Medicine  and  of  Clinical 
Medicine  in  the  Jefferson  Medical  College,  Philadelphia.  One  hand- 
some imperial  octavo  volume  of  1326  pages.  Illustrated.  Cloth, 
$7.00  net;  Sheep  or  Half  Morocco,  $8.00  net.  Sold  by  Subscription. 

"  As  a  work  either  for  study  or  reference  it  will  be  of  great  value  to  the  practitioner,  as 
it  is  virtually  an  exposition  of  such  clinical  therapeutics  as  experience  has  taught  to  be  oi 
the  most  value.  Taking  it  all  in  all,  no  recent  publication  on  therapeutics  can  be  compared 
with  this  one  in  practical  value  to  the  working  physician." — Chicago  Clinical  Review. 

"The  whole  field  of  medicine  has  been  well  covered.  The  work  is  thoroughly  prac- 
tical, and  while  it  is  intended  for  practitioners  and  students,  it  is  a  better  book  for  the  general 
practitioner  than  for  the  student.  The  young  practitioner  especially  will  find  it  extremely 
suggestive  and  helpful." — The  Indian  Lancet. 

AN  AMERICAN  TEXT-BOOK  OF  THE  DISEASES  OF  CHILDREN. 
Second  Edition,  Revised. 

By  65  Eminent  Contributors.  Edited  "by  Louis  STARR,  M.  D.,  Con- 
sulting Pediatrist  to  the  Maternity  Hospital,  etc.  ;  assisted  by  THOMP- 
SON S.  WESTCOTT,  M.  D.,  Attending  Physician  to  the  Dispensary 
for  Diseases  of  Children,  Hospital  of  the  University  of  Pennsyl- 
vania. In  one  handsome  imperial  octavo  volume  of  1244  pages, 
profusely  illustrated.  Cloth,  $7.00  net;  Sheep  or  Half  Morocco, 
$8.00  net.  Sold  by  Subscription. 

"This  is  far  and  away  the  best  text-book  on  children's  diseases  ever  published  in  the 
English  language,  and  is  certainly  the  one  which  is  best  adapted  to  American  readers. 
We  congratulate  the  editor  upon  the  result  of  his  work,  and  heartily  commend  it  to  the 
attention  of  every  student  and  practitioner." — American  Journal  of  the  Medical  Sciences. 

AN  AMERICAN  TEXT-BOOK  OF  DISEASES  OF  THE  EYE,  EAR, 
NOSE,  AND  THROAT. 

By  58  Prominent  Specialists.  Edited  by  G.  E.  DE  SCHWEINITZ,  M.D., 
Professor  of  Ophthalmology  in  the  Jefferson  Medical  College,  Phila- 
delphia ;  and  B.  ALEXANDER  RANDALL,  M.D.,  Professor  of  Diseases 
of  the  Ear  in  the  University  of  Pennsylvania.  Imperial  octavo,  1251 
pages  ;  766  illustrations,  59  of  them  in  colors.  Cloth,  $7.00  net ;  Sheep 
or  Half  Morocco,  $8.00  net.  Sold  by  Subscription. 

Illustrated  Catalogue  of  the  "American  Text-Books"  sent  free  upon  application. 


4  Medical  Publications  of  W.  B.  Saunders. 

AN  AMERICAN   TEXT-BOOK    OF   GENITO-URINARY  AND  SKIN 
DISEASES. 

By  47  Eminent  Specialists  and  Teachers.  Edited  by  L.  BOLTON 
BANGS,  M.  D.,  Professor  of  Genito- Urinary  Surgery,  University  and 
Bellevue  Hospital  Medical  College,  New  York  ;  and  W.  A.  HARD- 
AWAY,  M.  D.,  Professor  of  Diseases  of  the  Skin,  Missouri  Medical 
College.  Imperial  octavo  volume  of  1229  pages,  with  300  engravings 
and  20  full-page  colored  plates.  Cloth,  $7.00  net;  Sheep  or  Half 
Morocco,  $8.00  net.  Sold  by  Subscription. 

"  This  volume  is  one  of  the  best  yet  issued  of  the  publisher's  series  of  '  American  Text- 
Books.'  The  list  of  contributors  represents  an  extraordinary  array  of  talent  and  extended 
experience.  The  book  will  easily  take  the  place  in  comprehensiveness  and  value  of  the 
half  dozen  or  more  costly  works  on  these  subjects  which  have  heretofore  been  necessary  to 
a  well-equipped  library." — New  York  Polyclinic. 

AN  AMERICAN  TEXT-BOOK  OF  GYNECOLOQY,  MEDICAL  AND 
SURGICAL.     Second  Edition,  Revised. 

By  10  of  the  Leading  Gynecologists  of  America.  Edited  by  J.  M. 
BALDY,  M.  D.,  Professor  of  Gynecology  in  the  Philadelphia  Polyclinic, 
etc.  Handsome  imperial  octavo  volume  of  718  pages,  with  341  illus- 
trations in  the  text,  and  38  colored  and  half-tone  plates.  Cloth,  $6.00 
net;  Sheep  or  Half  Morocco,  $7.00  net.  Sold  by  Subscription. 

"  It  is  practical  from  beginning  to  end.  Its  descriptions  of  conditions,  its  recommen- 
dations for  treatment,  and  above  all  the  necessary  technique  of  different  operations,  are 
clearly  and  admirably  presented.  .  .  .  It  is  well  up  to  the  most  advanced  views  of  the 
day,  and  embodies  all  the  essential  points  of  advanced  American  gynecology.  It  is  destined 
to  make  and  hold  a  place  in  gynecological  literature  which  will  be  peculiarly  its  own." — 
Medical  Record,  New  York. 

AN  AMERICAN  TEXT-BOOK  OF  LEGAL  MEDICINE  AND  TOXI- 
COLOGY. 

Edited  by  FREDERICK  PETERSON,  M.D.,  Clinical  Professor  of  Mental 
Diseases  in  the  Woman's  Medical  College,  New  York  ;  Chief  of  Clinic, 
Nervous  Department,  College  of  Physicians  and  Surgeons,  New  York ; 
and  WALTER  S.  HAINES,  M.D.,  Professor  of  Chemistry,  Pharmacy, 
and  Toxicology  in  Rush  Medical  College,  Chicago.  In  Preparation. 

AN  AMERICAN  TEXT-BOOK  OF  OBSTETRICS. 

By  15  Eminent  American  Obstetricians.  Edited  by  RICHARD  C.  NOR- 
RIS,  M.D.;  Art  Editor,  ROBERT  L.  DICKINSON,  M.D.  One  handsome 
imperial  octavo  volume  of  1014  pages,  with  nearly  900  beautiful  colored 
and  half-tone  illustrations.  Cloth,  $7.00  net;  Sheep  or  Half  Morocco, 
$8.00  net.  Sold  by  Subscription. 

"  Permit  me  to  say  that  your  American  Text-Book  of  Obstetrics  is  the  most  magnificent 
medical  work  that  I  have  ever  seen.  I  congratulate  you  and  thank  you  for  this  superb  work, 
which  alone  is  sufficient  to  place  you  first  in  the  ranks  of  medical  publishers." — ALEXANDER 
J.  C.  SKENE,  Professor  of  Gynecology  in  the  Long  Island  College  Hospital,  Brooklyn,  N.  Y. 

"  This  is  the  most  sumptuously  illustrated  work  on  midwifery  that  has  yet  appeared.  In 
the  number,  the  excellence,  and  the  beauty  of  production  of  the  illustrations  it  far  surpasses 
every  other  book  upon  the  subject.  This  feature  alone  makes  it  a  work  which  no  medical 
library  should  omit  to  purchase." — British  Medical  Journal. 

"  As  an  authority,  as  a  book  of  reference,  as  a  '  working  book  '  for  the  student  or  prac- 
titioner, we  commend  it  because  we  believe  there  is  no  better." — American  Journal  of  the 
Medical  Sciences. 

Illustrated  Catalogue  of  the  "American  Text-Books  "  sent  free  upon  application. 


Medical  Publications  of  W.  B.  Saunders.  5 

AN  AMERICAN  TEXT=BOOK  OF  PATHOLOGY. 

Edited  by  JOHN  GUITERAS,  M.D.,  Professor  of  General  Pathology  and 
of  Morbid  Anatomy  in  the  University  of  Pennsylvania ;  and  DAVID 
RIESMAN,  M.D.,  Demonstrator  of  Pathological  Histology  in  the 
University  of  Pennsylvania.  In  Preparation. 

AN  AMERICAN  TEXT-BOOK  OF  PHYSIOLOGY. 

By  10  of  the  Leading  Physiologists  of  America.  Edited  by  WILLIAM 
H.  HOWELL,  PH.D.,  M.D.,  Professor  of  Physiology  in  the  Johns  Hop- 
kins University,  Baltimore,  Md.  One  handsome  imperial  octavo 
volume  of  1052  pages.  Illustrated.  Cloth,  $6.00  net;  Sheep  or  Half 
Morocco,  $7.00  net.  Sold  by  Subscription. 

"  We  can  commend  it  most  heartily,  not  only  to  all  students  of  physiology,  but  to  every 
physician  and  pathologist,  as  a  valuable  and  comprehensive  work  of  reference,  written  by 
men  who  are  of  eminent  authority  in  their  own  special  subjects." — London  Lancet. 

"  To  the  practitioner  of  medicine  and  to  the  advanced  student  this  volume  constitutes, 
we  believe,  the  best  exposition  of  the  present  status  of  the  science  of  physiology  in  the 
English  language." — American  Journal  of  the  Medical  Sciences. 

AN  AMERICAN  TEXT-BOOK  OF  SURGERY.     Second  Edition. 

By  13  Eminent  Professors  of  Surgery.  Edited  by  WILLIAM  W.  KEEN, 
M.D.,  LL.D.,  and  J.  WILLIAM  WHITE,  M.D.,  PH.D.  Handsome 
imperial  octavo  volume  of  1250  pages,  with  500  wood-cuts  in  the  text, 
and  39  colored  and  half-tone  plates.  Thoroughly  revised  and  enlarged, 
with  a  section  devoted  to  "  The  Use  of  the  Rontgen  Rays  in  Surgery." 
Cloth,  $7.00  net;  Sheep  or  Half  Morocco,  $8.00  net.  Sold  by  Sub- 
scription. 

«'  Personally,  I  should  not  mind  it  being  called  THE  TEXT-BOOK  (instead  of  A  TEXT- 
BOOK) ,  for  I  know  of  no  single  volume  which  contains  so  readable  and  complete  an  account 
of  the  science  and  art  of  Surgery  as  this  does." — EDMUND  OWEN,  F.R.C.S.,  Member  of 
the  Board  of  Examiners  of  the  Royal  College  of  Surgeons,  England. 

"  If  this  text-book  is  a  fair  reflex  of  the  present  position  of  American  surgery^  we  must 
admit  it  is  of  a  very  high  order  of  merit,  and  that  English  surgeons  will  have  to  look  very 
carefully  to  their  laurels  if  they  are  to  preserve  a  position  in  the  van  of  surgical  practice." — 
London  Lancet. 

AN  AMERICAN  TEXT-BOOK  OF  THE  THEORY  AND  PRACTICE 
OF  MEDICINE. 

By  12  Distinguished  American  Practitioners.  Edited  by  WILLIAM 
PEPPER,  M.D.,  LL.D.,  Professor  of  the  Theory  and  Practice  of  Medi- 
cine and  of  Clinical  Medicine  in  the  University  of  Pennsylvania.  Two 
handsome  imperial  octavo  volumes  of  about  1000  pages  each.  Illus- 
trated. Prices  per  volume  :  Cloth,  $5.00  net ;  Sheep  or  Half  Morocco, 
$6.00  net.  Sold  by  Subscription. 

"  I  am  quite  sure  it  will  commend  itself  both  to  practitioners  and  students  of  medicine, 
and  become  one  of  our  most  popular  text-books." — ALFRED  LOOMIS,  M.D.,  LL.D.,  Pro- 
fessor of  Pathology  and  Practice  of  Medicine,  University  of  the  City  of  New  York. 

"  We  reviewed  the  first  volume  of  this  work,  and  said  :  « It  is  undoubtedly  one  of  the 
best  text-books  on  the  practice  of  medicine  which  we  possess. '  A  consideration  of  the 
second  and  last  volume  leads  us  to  modify  that  verdict  and  to  say  that  the  completed  work 
is  in  our  opinion  the  best  si  its  kind  it  has  ever  been  our  fortune  to  see." — New  York  Medical 
Journal. 

Illustrated  Catalogue  of  the  "American  Text-Books "  sent  free  upon  application. 


6  Medical  Publications  of  W.  B.  Saunders. 

AN  AMERICAN  YEAR-BOOK  OF  MEDICINE  AND  SURGERY. 

A  Yearly  Digest  of  Scientific  Progress  and  Authoritative  Opinion  in  all 
branches  of  Medicine  and  Surgery,  drawn  from  journals,  monographs, 
and  text-books  of  the  leading  American  and  Foreign  authors  and 
investigators.  Collected  and  arranged,  with  critical  editorial  com- 
ments, by  eminent  American  specialists  and  teachers,  under  the  general 
editorial  charge  of  GEORGE  M.  GOULD,  M.D.  One  handsome  imperial 
octavo  volume  of  about  1200  pages.  Uniform  in  style,  size,  and 
general  make-up  with  the  "American  Text-Book"  Series.  Cloth, 
$6.50  net;  Half  Morocco,  $7.50  net.  Sold  by  Subscription. 

"  It  is  difficult  to  know  which  to  admire  most — the  research  and  industry  of  the  distin- 
guished band  of  experts  whom  Dr.  Gould  has  enlisted  in  the  service  of  the  Year-Book,  or  the 
wealth  and  abundance  of  the  contributions  to  every  department  of  science  that  have  been 
deemed  worthy  of  analysis.  .  .  .  It  is  much  more  than  a  mere  compilation  of  abstracts, 
for,  as  each  section  is  entrusted  to  experienced  and  able  contributors,  the  reader  has  the 
advantage  of  certain  critical  commentaries  and  expositions  .  .  .  proceeding  from  writers 
fully  qualified  to  perform  these  tasks.  .  .  .  It  is  emphatically  a  book  which  should  find 
a  place  in  every  medical  library,  and  is  in  several  respects  more  useful  than  the  famous 
'Jahrbucher'  of  Germany." — London  Lancet. 

THE  AMERICAN  POCKET  MEDICAL  DICTIONARY. 

[See  Dor  land's  Pocket  Dictionary,  page  10.] 

ANDERS'  PRACTICE  OF  MEDICINE.    Second  Edition. 

A  Text-Book  of  the  Practice  of  Medicine.  By  JAMES  M.  ANDERS, 
M.D.,  PH.D.,  LL.D.,  Professor  of  the  Practice  of  Medicine  and  of 
Clinical  Medicine,  Medico- Chirurgical  College,  Philadelphia.  In  one 
handsome  octavo  volume  of  1287  pages,  fully  illustrated.  Cloth, 
$5.50  net;  Sheep  or  Half  Morocco,  $6.50  net. 

"  It  is  an  excellent  book, — concise,  comprehensive,  thorough,  and  up  to  date.  It  is  a 
credit  to  you  ;  but,  more  than  that,  it  is  a  credit  to  the  profession  of  Philadelphia — to  us." 
JAMES  C.  WILSON,  Professor  of  the  Practice  of  Medicine  and  Clinical  Medicine,  Jefferson 
Medical  College,  Philadelphia. 

ASHTON'S  OBSTETRICS.     Fourth  Edition,  Revised. 

Essentials  of  Obstetrics.  By  W.  EASTERLY  ASHTON,  M.D.,  Pro. 
fessor  of  Gynecology  in  the  Medico-Chirurgical  College,  Philadelphia. 
Crown  octavo,  252  pages;  75  illustrations.  Cloth,  $1.00;  interleaved 
for  notes,  $1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"  Embodies  the  whole  subject  in  a  nut-shell.  We  cordially  recommend  it  to  our  read- 
ers."— Chicago  Medical  Times. 

BALL'S  BACTERIOLOGY.     Third  Edition,  Revised. 

Essentials  of  Bacteriology  ;  a  Concise  and  Systematic  Introduction 
to  the  Study  of  Micro-organisms.  By  M.  V.  BALL,  M.D.,  Bacteriol- 
ogist to  St.  Agnes'  Hospital,  Philadelphia,  etc.  Crown  octavo,  218 
pages;  82  illustrations,  some  in  colors,  and  5  plates.  Cloth,  $1.00; 
interleaved  for  notes,  11.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"  The  student  or  practitioner  can  readily  obtain  a  knowledge  of  the  subject  from  a  perusal 
of  this  book.  The  illustrations  are  clear  and  satisfactory." — Medical  Record,  New  York. 


Medical  Publications  of  W.  B.  Saunders.  7 

BASTIN'S  BOTANY. 

Laboratory  Exercises  in  Botany.  By  EDSON  S.  BASTIN,  M.A., 
late  Professor  of  Materia  Medica  and  Botany,  Philadelphia  College  of 
Pharmacy.  Octavo  volume  of  536  pages,  with  87  plates.  Cloth,  $2.50. 

"It  is  unquestionably  the  best  text-book  on  the  subject  that  has  yet  appeared.  The 
work  is  eminently  a  practical  one.  We  regard  the  issuance  of  this  book  as  an  important 
event  in  the  history  of  pharmaceutical  teaching  in  this  country,  and  predict  for  it  an  unquali- 
fied success." — Alumni  Report  to  the  Philadelphia  College  of  Pharmacy. 

' '  There  is  no  work  like  it  in  the  pharmaceutical  or  botanical  literature  of  this  country, 
and  we  predict  for  it  a  wide  circulation." — American  Journal  of  Pharmacy. 

BECK'S  SURGICAL  ASEPSIS. 

A  Manual  of  Surgical  Asepsis.  By  CARL  BECK,  M.D.,  Surgeon  to 
St.  Mark's  Hospital  and  the  New  York  German  Poliklinik,  etc.  306 
pages;  65  text-illustrations,  and  12  full- page  plates.  Cloth,  $1.25  net. 

"  An  excellent  exposition  of  the  '  very  latest'  in  the  treatment  of  wounds  as  practised 
by  leading  German  and  American  surgeons." — Birmingham  (Eng.)  Medical  Review. 

"This  little  volume  can  be  recommended  to  any  who  are  desirous  of  learning  the  details 
of  asepsis  in  surgery,  for  it  will  serve  as  a  trustworthy  guide." — London  Lancet. 

BOISLINIERE'S  OBSTETRIC  ACCIDENTS,  EMERGENCIES,  AND 

OPERATIONS. 
Obstetric  Accidents,  Emergencies,  and  Operations.     By  L.  CH. 

BOISLINIERE,  M.D.,  late  Emeritus  Professor  of  Obstetrics,  St.  Louis 
Medical  College.  381  pages,  handsomely  illustrated.  Cloth,  $2.00  net. 

"It  is  clearly  and  concisely  written,  and  is  evidently  the  work  of  a  teacher  and  practi- 
tioner of  large  experience." — British  Medical  Journal. 

"  A  manual  so  useful  to  the  student  or  the  general  practitioner  has  not  been  brought  to 
our  notice  in  a  long  time.  The  field  embraced  in  the  title  is  covered  in  a  terse,  interesting 
way." —  Yale  Medical  Journal. 

BROCKWAY'S  MEDICAL  PHYSICS.     Second  Edition,  Revised. 
Essentials  of   Medical   Physics.     By  FRED  J.  BROCKWAY,  M.D., 
Assistant  Demonstrator  of  Anatomy  in  the  College  of  Physicians  and 
Surgeons,  New  York.     Crown  octavo,  330  pages ;   155  fine  illustrations. 
Cloth,  $1.00  net ;  interleaved  for  notes,  $1.25  net. 

[See  Saunders'  Question- Compends,  page  21.] 

"  The  student  who  is  well  versed  in  these  pages  will  certainly  prove  qualified  to  com- 
prebend  with  ease  and  pleasure  the  great  majority  of  questions  involving  physical  principles 
likely  to  be  met  with  in  his  medical  studies." — American  Practitioner  and  News. 

"We  know  of  no  manual  that  affords  the  medical  student  a  better  or  more  concise 
exposition  of  physics,  and  the  book  may  be  commended  as  a  most  satisfactory  presentation 
of  those  essentials  that  are  requisite  in  a  course  in  medicine." — New  York  Medical  Journal. 

"  It  contains  all  that  one  need  know  on  the  subject,  is  well  written,  and  is  copiously 
illustrated." — Medical  Record,  New  York. 

BURR  ON  NERVOUS  DISEASES. 

A  Manual  of  Nervous  Diseases.  By  CHARLES  W.  BURR,  M.D., 
Clinical  Professor  of  Nervous  Diseases,  Medico-Chirurgical  College, 
Philadelphia ;  Pathologist  to  the  Orthopedic  Hospital  and  Infirmary 
for  Nervous  Diseases;  Visiting  Physician  to  St.  Joseph's  Hospital,  etc. 
In  Preparation. 


8  Medical  Publications  of  W.  B.  Saundcrs. 

BUTLER'S  MATERIA    MEDICA,   THERAPEUTICS,   AND   PHAR- 
MACOLOGY.    Second  Edition,  Revised. 

A  Text-Book  of  Materia  Medica,  Therapeutics,  and  Pharma- 
cology. By  GEORGE  F.  BUTLER,  PH.G.,  M.D.,  Professor  of  Materia 
Medica  and  of  Clinical  Medicine  in  the  College  of  Physicians  and 
Surgeons,  Chicago ;  Professor  of  Materia  Medica  and  Therapeutics, 
Northwestern  University,  Woman's  Medical  School,  etc.  Octavo,  860 
pages,  illustrated.  Cloth,  $4.00  net;  Sheep,  $5.00  net. 

"  Taken  as  a  whole,  the  book  may  fairly  be  considered  as  one  of  the  most  satisfactory 
of  any  single-volume  works  on  materia  medica  in  the  market," — Journal  of  the  American 
Medical  Association. 

CERNA  ON  THE  NEWER  REMEDIES.  Second  Edition,  Revised. 
Notes  on  the  Newer  Remedies,  their  Therapeutic  Applications 
and  Modes  of  Administration.  By  DAVID  CERNA,  M.D.,  PH.D., 
formerly  Demonstrator  of  and  Lecturer  on  Experimental  Therapeutics 
in  the  University  of  Pennsylvania ;  Demonstrator  of  Physiology  in  the 
Medical  Department  of  the  University  of  Texas.  Rewritten  and 
greatly  enlarged.  Post-octavo,  253  pages.  Cloth,  $1.25. 

"The  appearance  of  this  new  edition  of  Dr.  Cerna's  very  valuable  work  shows  that  it 
is  properly  appreciated.  The  book  ought  to  be  in  the  possession  of  every  practising  physi- 
cian."— New  York  Medical  Journal. 

CHAPIN  ON  INSANITY. 

A  Compendium  of  Insanity.     By  JOHN  B.  CHAPIN,  M.D.,  LL.D., 

Physician-in-Chief,  Pennsylvania  Hospital  for  the  Insane ;  late  Physi- 
cian-Superintendent of  the  Willard  State  Hospital,  New  York ;  Hon- 
orary Member  of  the  Medico-Psychological  Society  of  Great  Britain, 
of  the  Society  of  Mental  Medicine  of  Belgium.  i2ino,  234  pages, 
illustrated.  Cloth,  $1.25  net. 

"  The  practical  parts  of  Dr.  Chapin's  book  are  what  constitute  its  distinctive  merit.    We 
desire  especially  to  call  attention  to  the  fact  that  on  the  subject  of  therapeutics  of  insanity 
the  work  is  exceedingly  valuable.     It  is  not  a  made  book,  but  a  genuine  condensed  thesis, 
which  has  all  the  value  of  ripe  opinion  and  all  the  charm  of  a  vigorous  and  natural  style. "- 
Philadelphia  Medical  Journal. 

CHAPMAN'S    MEDICAL   JURISPRUDENCE    AND    TOXICOLOGY. 

Second  Edition,  Revised. 

Medical  Jurisprudence  and  Toxicology.  By  HENRY  C.  CHAPMAN, 
M.D.,  Professor  of  Institutes  of  Medicine  and  Medical  Jurisprudence 
in  the  Jefferson  Medical  College  of  Philadelphia.  254  pages,  with  55 
illustrations  and  3  full-page  plates  in  colors.  Cloth,  $1.50  net. 

"The  best  book  of  its  class  for  the  undergraduate  that  we  know  of." — New  York 
Medical  Times. 

CHURCH  AND  PETERSON'S  NERVOUS  AND  MENTAL  DISEASES. 
Nervous  and  Mental  Diseases.  By  ARCHIBALD  CHURCH,  M.  I)., 
Professor  of  Mental  Diseases  and  Medical  Jurisprudence  in  the  North- 
western University  Medical  School,  Chicago;  and  FRI  I>I:KK  K  Ti  i  IK- 
SON,  M.  D.,  Clinical  Professor  of  Mental  Diseases,  Woman's  Medical 
College,  N.  Y.;  Chief  of  Clinic,  Nervous  ])ept.,  College  of  Physi- 
cians and  Surgeons,  N.  Y.  Handsome  octavo  volume  of  843  pages, 
profusely  illustrated.  Cloth,  $5.00  net;  Half  Morocco,  56.00  net. 


Medical  Publications  of  W.  B.  Saunders.  9 

CLARKSON'S  HISTOLOGY. 

A   Text=Book    of    Histology,    Descriptive   and    Practical.      By 

ARTHUR  CLARKSON,  M.B.,  C.M.  Edin.,  formerly  Demonstrator  of 
Physiology  in  the  Owen's  College,  Manchester;  late  Demonstrator  of 
Physiology  in  Yorkshire  College,  Leeds.  Large  octavo,  554  pages; 
22  engravings  in  the  text,  and  174  beautifully  colored  original  illustra- 
tions. Cloth,  strongly  bound,  $6.00  net. 

"The  work  must  be  considered  a  valuable  addition  to  the  list  of  available  text- books, 
and  is  to  be  highly  recommended." — New  York  Medical  Journal. 

"This  is  one  of  the  best  works  for  students  we  have  ever  noticed.  We  predict  that  the 
book  will  attain  a  well -deserved  popularity  among  our  students." — Chicago  Medical  Recorder. 

CLIMATOLOGY. 

Transactions  of  the  Eighth  Annual  Meeting  of  the  American 
Climatological  Association,  held  in  Washington,  September  22-25, 
1891.  Forming  a  handsome  octavo  volume  of  276  pages,  uniform  with 
remainder  of  series.  (A  limited  quantity  only.)  Cloth,  $1.50. 

COHEN  AND  ESHNER'S  DIAGNOSIS. 

Essentials  of  Diagnosis.  By  SOLOMON  SOLIS-COHEN,  M.D.,  Pro- 
fessor of  Clinical  Medicine  and  Applied  Therapeutics  in  the  Philadel- 
phia Polyclinic  ;  and  AUGUSTUS  A.  ESHNER,  M.D.,  Professor  of  Clinical 
Medicine  in  the  Philadelphia  Polyclinic.  Post-octavo,  382  pages;  55 
illustrations.  Cloth,  $1.50  net. 

[See  Saunders    Question-  Compends,  page  21.] 

"We  can  heartily  commend  the  book  to  all  those  who  contemplate  purchasing  a  <com- 
pend.'  It  is  modern  and  complete,  and  will  give  more  satisfaction  than  many  other  works 
which  are  perhaps  too  prolix  as  well  as  behind  the  times." — Medical  Review,  St.  Louis. 

CORWIN'S  PHYSICAL  DIAGNOSIS. 

Essentials  of  Physical  Diagnosis  of  the  Thorax.  By  ARTHUR 
M.  CORWIN,  A.M.,  M.D.,  Demonstrator  of  Physical  Diagnosis  in  Rush 
Medical  College,  Chicago ;  Attending  Physician  to  Central  Free  Dis- 
pensary, Department  of  Rhinology,  Laryngology,  and  Diseases  of  the 
Chest,  Chicago.  200  pages,  illustrated.  Cloth,  flexible  covers,  $1.25  net. 

"It  is  excellent.  The  student  who  shall  use  it  as  his  guide  to  the  careful  study  of 
physical  exploration  upon  normal  and  abnormal  subjects  can  scarcely  fail  to  acquire  a  good 
working  knowledge  of  the  subject." — Philadelphia  Polyclinic. 

"A  most  excellent  little  work.  It  brightens  the  memory  of  the  differential  diagnostic 
signs,  and  it  arranges  orderly  and  in  sequence  the  various  objective  phenomena  to  logical 
solution  of  a  careful  diagnosis. " — Journal  of  Nervous  and  Mental  Diseases. 

CRAGIN'S  GYNAECOLOGY.     Fourth  Edition,  Revised. 

Essentials  of  Gynaecology.  By  EDWIN  B.  CRAGIN,  M.  D.,  Lecturer 
in  Obstetrics,  College  of  Physicians  and  Surgeons,  New  York.  Crown 
octavo,  200  pages;  62  illustrations.  Cloth,  $1.00  ;  interleaved  for  notes, 
$1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"  A  handy  volume,  and  a  distinct  improvement  on  students'  compends  in  general.  No 
author  who  was  not  himself  a  practical  gynecologist  could  have  consulted  the  student's  needs 
so  thoroughly  as  Dr.  Cragin  has  done." — Medical  Record,  New  York. 


10  Medical  Publications  of  W.  B.  Saunders. 

CROOKSHANK'S  BACTERIOLOGY.     Fourth  Edition,  Revised. 

A  Text-Book  of  Bacteriology.  By  EDGAR  M.  CROOKSHANK,  M.B., 
Professor  of  Comparative  Pathology  and  Bacteriology,  King's  College, 
London.  Octavo  volume  of  700  pages,  with  273  engravings  and  22 
original  colored  plates.  Cloth,  $6.50  net;  Half  Morocco,  $7.50  net. 

"  To  the  student  who  wishes  to  obtain  a  good  resume  of  what  has  been  done  in  bacteri- 
ology, or  who  wishes  an  accurate  account  of  the  various  methods  of  research,  the  book  may 
be  recommended  with  confidence  that  he  will  find  there  what  he  requires." — London  Lancet. 

DACOSTA'S  SURGERY.  Second  Ed.,  Revised  and  Greatly  Enlarged. 
Modern  Surgery,  General  and  Operative.  By  JOHN  CHALMERS 
DACOSTA,  M.D.,  Clinical  Professor  of  Surgery,  Jefferson  Medical 
College,  Philadelphia;  Surgeon  to  the  Philadelphia  Hospital,  etc. 
Handsome  octavo  volume  of  900  pages,  profusely  illustrated.  Cloth, 
$4.00  net;  Half  Morocco,  $5.00  net. 

"We  know  of  no  small  work  on  surgery  in  the  English  language  which  so  well  fulfils 
the  requirements  of  the  modern  student." — Medico-Chirurgical  Journal,  Bristol,  England. 

DE  SCHWEINITZ  ON  DISEASES  OF  THE  EYE.      Third  Edition, 

Revised. 
Diseases  of   the  Eye,     A  Handbook   of   Ophthalmic   Practice. 

By  G.  E.  DE  SCHWEINITZ,  M.D.,  Professor  of  Ophthalmology  in  the 
Jefferson  Medical  College,  Philadelphia,  etc.  Handsome  royal  octavo 
volume  of  696  pages,  with  256  fine  illustrations  and  2  chromo-litho- 
graphic  plates.  Cloth,  $4.00  net ;  Sheep  or  Half  Morocco,  $5.00  net. 

"  A  clearly  written,  comprehensive  manual.  One  which  we  can  commend  to  students 
as  a  reliable  text-book,  written  with  an  evident  knowledge  of  the  wants  of  those  entering 
upon  the  study  of  this  special  branch  of  medical  science." — British  Medical  Journal. 

"A  work  that  will  meet  the  requirements  not  only  of  the  specialist,  but  of  the  general 
practitioner  in  a  rare  degree.  I  am  satisfied  that  unusual  success  awaits  it." — WILLIAM 
PEPPER,  M.D.,  Professor  of  the  Theory  and  Practice  of  Medicine  and  Clinical  Medicine, 
University  of  Pennsylvania. 

DORLAND'S  DICTIONARY.     Second  Edition,  Revised. 

'  The  American  Pocket  Medical  Dictionary.  Containing  the  Pro- 
nunciation and  Definition  of  over  26,000  words  and  phrases,  and  a  large 
number  of  useful  tables.  Edited  by  W.  A.  NEWMAN  BORLAND,  M.  I)., 
Assistant  Demonstrator  of  Obstetrics,  University  of  Pennsylvania  ;  Fel- 
low of  the  American  Academy  of  Medicine.  518  pages;  handsomely 
bound  in  full  leather,  limp,  with  gilt  edges  and  patent  index.  Price, 
$1.25  net. 

DORLAND'S  OBSTETRICS. 

A  Manual  of  Obstetrics.  By  W.  A.  NEWMAN  DORLAND,  M.D., 
Assistant  Demonstrator  of  Obstetrics,  University  of  Pennsylvania; 
Instructor  in  Gynecology  in  the  Philadelphia  Polyclinic.  760  pages; 
163  illustrations  in  the  text,  and  6  full-page  plates.  Cloth,  $2.50  net. 

"  By  far  the  best  book  on  this  subject  that  has  ever  come  to  our  notice." — American 
Medical  Review. 

"  It  has  rarely  been  our  duty  to  review  a  book  which  lias  trivm  us  more  pleasure  in  its 
perusal  and  more  satisfaction  in  its  criticism.  It  is  a  veritable  encyclopedia  of  knowledge, 
a  gold  mine  of  practical,  concise  thoughts." — American  Medico-Surgical  Bulletin. 


Medical  Publications  of  W.  B.  Saunders.  11 

FROTHINGHAM'S  GUIDE  FOR  THE  BACTERIOLOGIST. 

Laboratory  Guide  for  the  Bacteriologist.  By  LANGDON  FROTH- 
INGHAM,  M.D.V.,  Assistant  in  Bacteriology  and  Veterinary  Science, 
Sheffield  Scientific  School,  Yale  University.  Illustrated.  Cloth,  75  cts. 

"  It  is  a  convenient  and  useful  little  work,  and  will  more  than  repay  the  outlay  neces- 
sary for  its  purchase  in  the  saving  of  time  which  would  otherwise  be  consumed  in  looking 
up  the  various  points  of  technique  so  clearly  and  concisely  laid  down  in  its  pages." — Ameri- 
can Medico- Surgical  Bulletin. 

GARRIGUES'  DISEASES  OF  WOMEN.  Second  Edition,  Revised. 
Diseases  of  Women.  By  HENRY  J.  GARRIGUES,  A.M.,  M.D.,  Pro- 
fessor of  Gynecology  in  the  New  York  School  of  Clinical  Medicine ; 
Gynecologist  to  St.  Mark's  Hospital  and  to  the  German  Dispensary, 
New  York  City,  etc.  Handsome  octavo  volume  of  728  pages,  illus- 
trated by  335  engravings  and  colored  plates.  Cloth,  $4.00  net; 
Sheep  or  Half  Morocco,  $5.00  net. 

"  One  of  the  best  text-books  for  students  and  practitioners  which  has  been  published  in 
the  English  language  ;  it  is  condensed,  clear,  and  comprehensive.  The  profound  learning 
and  great  clinical  experience  of  the  distinguished  author  find  expression  in  this  book  in  a 
most  attractive  and  instructive  form.  Young  practitioners  to  whom  experienced  consultants 
may  not  be  available  will  find  in  this  book  invaluable  counsel  and  help." — THAD.  A. 
REAMY,  M.D.,  LL.D.,  Professor  of  Clinical  Gynecology,  Medical  College  of  Ohio. 

GLEASON'S  DISEASES  OF  THE  EAR.  Second  Edition,  Revised. 
Essentials  of  Diseases  of  the  Ear.  By  E.  B.  GLEASON,  S.B., 
M.D. ,  Clinical  Professor  of  Otology,  Medico-Chirurgical  College, 
Philadelphia  ;  Surgeon -in -Charge  of  the  Nose,  Throat,  and  Ear  Depart- 
ment of  the  Northern  Dispensary,  Philadelphia.  208  pages,  with 
114  illustrations.  Cloth,  $1.00  ;  interleaved  for  notes,  $1.25. 
[See  Saunders'  Question- Compends,  page  21.] 

"  It  is  just  the  book  to  put  into  the  hands  of  a  student,  and  cannot  fail  to  give  him  a 
useful  introduction  to  ear-affections  ;  while  the  style  of  question  and  answer  which  is  adopted 
throughout  the  book  is,  we  believe,  the  best  method  of  impressing  facts  permanently  on  the 
mind. " — Liverpool  Medico-  Chirurgical  Journal. 

GOULD  AND  PYLE'S  CURIOSITIES  OF  MEDICINE. 

Anomalies  and  Curiosities  of  Medicine.  By  GEORGE  M.  GOULD, 
M.D.,  and  WALTER  L.  PYLE,  M.D.  An  encyclopedic  collection  of 
rare  and  extraordinary  cases  and  of  the  most  striking  instances  of 
abnormality  in  all  branches  of  Medicine  and  Surgery,  derived  from  an 
exhaustive  research  of  medical  literature  from  its  origin  to  the  present 
day,  abstracted,  classified,  annotated,  and  indexed.  Handsome  im- 
perial octavo  volume  of  968  pages,  with  295  engravings  in  the  text, 
and  12  full-page  plates.  Cloth,  $6.00  net;  Half  Morocco,  $7.00  net. 
Sold  by  Subscription. 

"  One  of  the  most  valuable  contributions  ever  made  to  medical  literature.  It  is,  so  far 
as  we  know,  absolutely  unique,  and  every  page  is  as  fascinating  as  a  novel.  Not  alone  for 
the  medical  profession  has  this  volume  value:  it  will  serve  as  a  book  of  reference  for  all  who 
are  interested  in  general  scientific,  sociologic,  or  medico-legal  topics." — Brooklyn  Medical 
Journal. 

"This  is  certainly  a  most  remarkable  and  interesting  volume.  It  stands  alone  among 
medical  literature,  an  anomaly  on  anomalies,  in  that  there  is  nothing  like  it  elsewhere  in 
medical  literature.  It  is  a  book  full  of  revelations  from  its  first  to  its  last  page,  and  cannot 
but  interest  and  sometimes  almost  horrify  its  readers." — American  Medico- Surgical  Bulletin. 


12  Medical  Publications  of  W.  B.  Saunders. 

GRAFSTROM'S   MECHANO-THERAPY. 

A  Text-Book  of  Mechano-Therapy  (Massage  and  Medical  Gym- 
nastics). By  AXEL  V.  GRAFSTROM,  B.  Sc.,  M.  D.,  late  Lieutenant  in 
the  Royal  Swedish  Army ;  late  House  Physician  City  Hospital,  Black- 
well's  Island,  New  York.  12010,  139  pages,  illustrated.  Cloth,  $1.00  net. 

GRIFFITH  ON  THE  BABY.     Second  Edition,  Revised. 

The  Care  of  the  Baby.  By  J.  P.  CROZER  GRIFFITH,  M.D.,  Clini- 
cal Professor  of  Diseases  of  Children,  University  of  Pennsylvania ; 
Physician  to  the  Children's  Hospital,  Philadelphia,  etc.  i2mo,  404 
pages,  with  67  illustrations  in  the  text,  and  5  plates.  Cloth,  $1.50. 

"  The  best  book  for  the  use  of  the  young  mother  with  which  we  are  acquainted.  .  .  . 
There  are  very  few  general  practitioners  who  could  not  read  the  book  through  with  advan- 
tage. ' ' — Archives  of  Pediatrics. 

"The  whole  book  is  characterized  by  rare  good  sense,  and  is  evidently  written  by  a 
master  hand.  It  can  be  read  with  benefit  not  only  by  mothers  but  by  medical  students  and 
by  any  practitioners  who  have  not  had  large  opportunities  for  observing  children." — Ameri- 


7, 


can  Journal  of  Obstetrics. 

GRIFFITH'S  WEIGHT  CHART. 

Infant's  Weight  Chart.  Designed  by  J.  P.  CROZER  GRIFFITH,  M.  D. , 
Clinical  Professor  of  Diseases  of  Children  in  the  University  of  Penn- 
sylvania, etc.  25  charts  in  each  pad.  Per  pad,  50  cents  net. 

A  convenient  blank  for  keeping  a  record  of  the  child's  weight  during  the  first  two  years 
of  life.  Printed  on  each  chart  is  a  curve  representing  the  average  weight  of  a  healthy  infant, 
so  that  any  deviation  from  the  normal  can  readily  be  detected. 

GROSS,  SAMUEL  D.,  AUTOBIOGRAPHY  OF. 

Autobiography  of  Samuel  D.  Gross,  M.D.,  Emeritus  Professor  of 
Surgery  in  the  Jefferson  Medical  College,  Philadelphia,  with  Remi- 
niscences of  His  Times  and  Contemporaries.  Edited  by  his  Sons, 
SAMUEL  W.  GROSS,  M.D.,.LL.D.,  late  Professor  of  Principles  of  Sur- 
gery and  of  Clinical  Surgery  in  the  Jefferson  Medical  College,  and 
A.  HALLER  GROSS,  A.M.,  of  the  Philadelphia  Bar.  Preceded  by  a 
Memoir  of  Dr.  Gross,  by  the  late  AUSTIN  FLINT,  M.D.,  LL.D.  In 
two  handsome  volumes,  each  containing  over  400  pages,  demy  octavo, 
extra  cloth,  gilt  tops,  with  fine  Frontispiece  engraved  on  steel.  Price 
per  volume,  $2.50  net. 

"Dr.  Gross  was  perhaps  the  most  eminent  exponent  of  medical  science  that  America 
has  yet  produced.  His  Autobiography,  related  as  it  is  with  a  fulness  and  completeness 
seldom  to  be  found  in  such  works,  is  an  interesting  and  valuable  book.  He  comments  on 
many  things,  especially,  of  course,  on  medical  men  and  medical  practice,  in  a  very  interest- 
ing way." — The  Spectator,  London,  England. 

HAMPTON'S  NURSING.  Second  Edition,  Revised  and  Enlarged. 
Nursing:  Its  Principles  and  Practice.  By  ISABEL  ADAMS  H AMP- 
TON,  Graduate  of  the  New  York  Training  School  for  Nurses  attached 
to  Bellevue  Hospital ;  late  Superintendent  of  Nurses  and  Principal  of 
the  Training  School  for  Nurses,  Johns  Hopkins  Hospital,  Baltimore, 
Md.  12  mo,  512  pages,  illustrated.  Cloth,  $2.00  net. 

"  Seldom  have  we  perused  a  book  upon  the  subject  that  has  given  us  so  much  pleasure 
as  the  one  before  us.  We  would  strongly  ur^c  upon  the  members  of  our  own  profession  the 
need  of  a  book  like  this,  for  it  will  enable  each  of  us  to  become  a  training  school  in  him- 
self.' ' —  Ontario  Medical  Jon  r>ial. 


Medical  Publications  of  W.  B.  Saunders.  13 

HARE'S  PHYSIOLOGY.  Fourth  Edition,  Revised. 

Essentials  of  Physiology.  By  H.  A.  HARE,  M.D.,  Professor  of 
Therapeutics  and  Materia  Medica  in  the  Jefferson  Medical  College  of 
Philadelphia.  Crown  octavo,  239  pages.  Cloth,  $1.00  net;  inter- 
leaved for  notes,  $1.25  net. 

[See  Saunders'  Question- Compends,  page  21.] 

"  The  best  condensation  of  physiological  knowledge  we  have  yet  seen." — Medicai 
Record,  New  York. 

HART'S  DIET  IN  SICKNESS  AND  IN  HEALTH. 

Diet  in  Sickness  and  in  Health.  By  MRS.  ERNEST  HART,  formerly 
Student  of  the  Faculty  of  Medicine  of  Paris  and  of  the  London  School 
of  Medicine  for  Women ;  with  an  INTRODUCTION  by  SIR  HENRY 
THOMPSON,  F.R.C.S.,  M.D.,  London.  220  pages.  Cloth,  $1.50. 

"  We  recommend  it  cordially  to  the  attention  of  all  practitioners ;  both  to  them  and  to 
their  patients  it  may  be  of  the  greatest  service." — New  York  Medical  Journal. 

HAYNES'  ANATOMY. 

A  Manual  of  Anatomy.  By  IRVING  S.  HAYNES,  M.D.,  Adjunct 
Professor  of  Anatomy  and  Demonstrator  of  Anatomy,  Medical  Depart- 
ment of  the  New  York  University,  etc.  680  pages,  illustrated  with  42 
diagrams  in  the  text,  and  134  full-page  half-tone  illustrations  from 
original  photographs  of  the  author's  dissections.  Cloth,  $2.50  net. 

"  This  book  is  the  work  of  a  practical  instructor — one  who  knows  by  experience  the 
requirements  of  the  average  student,  and  is  able  to  meet  these  requirements  in  a  very  satis- 
factory way.  The  book  is  one  that  can  be  commended." — Medical  Record,  New  York. 

HEISLER'S  EMBRYOLOGY. 

A  Text-Book  of  Embryology.  By  JOHN  C.  HEISLER,  M.D.,  Pro- 
fessor of  Anatomy  in  the  Medico- Chirurgical  College,  Philadelphia. 
i2mo  volume  of  about  325  pages,  handsomely  illustrated. 

HIRST'S  OBSTETRICS. 

A  Text- Book  of  Obstetrics.  By  BARTON  COOKE  HIRST,  M.  D., 
Professor  of  Obstetrics  in  the  University  of  Pennsylvania.  Handsome 
octavo  volume  of  848  pages,  with  618  illustrations,  and  7  colored 
plates.  Cloth,  $5.00  net;  Sheep  or  Half  Morocco,  $6.00  net. 

"  The  illustrations  are  numerous  and  are  works  of  art,  many  of  them  appearing  for  the 
first  time.  The  arrangement  of  the  subject-matter,  the  foot-notes,  and  index  are  beyond 
criticism.  As  a  true  model  of  what  a  modern  text-book  on  obstetrics  should  be,  we  feel 
justified  in  affirming  that  Dr.  Hirst's  book  is  without  a  rival." — New  York  Medical  Record. 

HYDE  AND  MONTGOMERY  ON  SYPHILIS  AND  THE  VENEREAL 

DISEASES. 

Syphilis  and  the  Venereal  Diseases.  By  JAMES  NEVINS  HYDE, 
M.D.,  Professor  of  Skin  and  Venereal  Diseases,  and  FRANK  H.  MONT- 
GOMERY, M.D.,  Lecturer  on  Dermatology  and  Genito-Urinary  Diseases 
in  Rush  Medical  College,  Chicago,  111.  618  pages,  profusely  illustrated. 
Cloth,  $2.50  net. 

"  We  can  commend  this  manual  to  the  student  as  a  help  to  him  in  his  study  of  venereal 
diseases. ' ' — Liverpool  Medico-  Chirurgical  Journal. 

"The  best  student's  manual  which  has  appeared  on  the  subject." — St.  Louis  Medical 
and  Surgical  Journal. 


14  Medical  Publications  of  W.  B.  Saunders. 

JACKSON  AND  GLEASON'S  DISEASES  OF  THE  EYE,  NOSE,  AND 

THROAT.     Second  Edition,  Revised. 

Essentials  of  Refraction  and  Diseases  of  the  Eye.  By  EDWARD 
JACKSON,  A.M.,  M.D.,  Professor  of  Diseases  of  the  Eye  in  the  Phila- 
delphia Polyclinic  and  College  for  Graduates  in  Medicine  ;  and — 
Essentials  of  Diseases  of  the  Nose  and  Throat.  By  E.  BALD- 
WIN GLEASON,  M.D.,  Surgeon-in-Charge  of  the  Nose,  Throat,  and 
Ear  Department  of  the  Northern  Dispensary  of  Philadelphia.  Two 
volumes  in  one.  Crown  octavo,  290  pages;  124  illustrations.  Cloth, 
$1.00;  interleaved  for  notes,  $1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"  Of  great  value  to  the  beginner  in  these  branches.  The  authors  are  both  capable  men, 
and  know  what  a  student  most  needs." — Medical  Record,  New  York. 

KEATING'S  DICTIONARY.     Second  Edition,  Revised. 

A  New  Pronouncing  Dictionary  of  Medicine,  with  Phonetic 
Pronunciation,  Accentuation,  Etymology,  etc.  BY  JOHN  M. 
KEATING,  M.D.,  LL.D.,  Fellow  of  the  College  of  Physicians  of  Phila- 
delphia; Vice-President  of  the  American  Psediatric  Society;  Editor 
"  Cyclopaedia  of  the  Diseases  of  Children,"  etc.;  and  HENRY 
HAMILTON,  Author  of  "A  New  Translation  of  Virgil's  ^Eneid  into 
English  Rhyme,"  etc.;  with  the  collaboration  of  J.  CHALMERS  DA- 
COSTA,  M.D.,  and  FREDERICK  A.  PACKARD,  M.D.  With  an  Appendix 
containing  Tables  of  Bacilli,  Micrococci,  Leucomames,  Ptomaines; 
Drugs  and  Materials  used  in  Antiseptic  Surgery;  Poisons  and  their 
Antidotes ;  Weights  and  Measures ;  Thermometric  Scales ;  New 
Official  and  Unofficial  Drugs,  etc.  One  volume  of  over  800  pages. 
Prices,  with  Denison's  Patent  Ready-Reference  Index:  Cloth,  $5.00 
net;  Sheep  or  Half  Morocco,  $6.00  net;  Half  Russia,  $6.50  net. 
Without  Patent  Index:  Cloth,  $4.00  net;  Sheep  or  Half  Morocco, 
$5.00  net. 

"  I  am  much  pleased  with  Keating's  Dictionary,  and  shall  take  pleasure  in  recommend- 
ing  it  to  my  classes." — HENRY  M.  LYMAN,  M.D.,  Professor  of  the  Principles  and  Practice 
of  Medicine,  Rush  Medical  College,  Chicago,  III. 

"  I  am  convinced  that  it  will  be  a  very  valuable  adjunct  to  my  study-table,  convenient 
in  size  and  sufficiently  full  for  ordinary  use." — C.  A.  LINDSLEY,  M.D.,  Professor  of  the 
Theory  and  Practice  of  Medicine,  Medical  Dept.  Yale  University. 

KEATING'S  LIFE  INSURANCE. 

How  to  Examine  for  Life  Insurance.  By  JOHN  M.  KEATING, 
M.D.,  Fellow  of  the  College  of  Physicians  of  Philadelphia;  Vice- 
President  of  the  American  Paediatric  Society;  Ex-President  of  the 
Association  of  Life  Insurance  Medical  Directors.  Royal  octavo,  211 
pages ;  with  two  large  half-tone  illustrations,  and  a  plate  prepared  by 
Dr.  McClellan  from  special  dissections ;  also,  numerous  other  illustra- 
tions. Cloth,  $2.00  net. 

"  This  is  by  far  the  riost  useful  book  which  has  yet  appeared  on  insurance  examination', 
a  subject  of  growing  interest  and  importance.  Not  the  least  valuable  portion  of  the  volume 
is  Part  II,  which  consists  of  instructions  issued  to  their  examining  physicians  by  twenty  tour 
representative  companies  of  this  country.  If  for  these  alone,  the  book  should  be  at  the  right 
hand  of  every  physician  interested  in  this  special  branch  of  medical  science." — The  Medical 
News. 


Medical  Publications  of  W.  B.  Saundcrs.  15 


KEEN  ON  THE  SURGERY  OF  TYPHOID  FEVER. 

The   Surgical  Complications  and  Sequels  of  Typhoid    Fever. 

By  WM.  W.  KEEN,  M.D.,  LL.D.,  Professor  of  the  Principles  of  Sur- 
gery and  of  Clinical  Surgery,  Jefferson  Medical  College,  Philadelphia; 
Corresponding  Member  of  the  Societe  de  Chirurgie,  Paris ;  Honorary 
Member  of  the  Societe  Beige  de  Chirurgie,  etc.  Octavo  volume  of 
386  pages,  illustrated.  Cloth,  $3.00  net. 

"  This  is  probably  the  first  and  only  work  in  the  English  language  that  gives  the  reader 
a  clear  view  of  what  typhoid  fever  really  is,  and  what  it  does  and  can  do  to  the  human 

organism.     This  book  should  be  in  the  possession  of  every  medical  man  in  America." 

American  Medico-Surgical  Bulletin, 

KEEN'S  OPERATION  BLANK.  Second  Edition,  Revised  Form. 
An  Operation  Blank,  with  Lists  of  Instruments,  etc.  Required 
in  Various  Operations.  Prepared  by  W.  W.  KEEN,  M.D.,  LL.D., 
Professor  of  the  Principles  of  Surgery  in  Jefferson  Medical  College, 
Philadelphia.  Price  per  pad,  containing  blanks  for  fifty  operations, 
50  cents  net. 

KYLE  ON  THE  NOSE  AND  THROAT. 

Diseases  of  the  Nose  and  Throat.  By  D.  BRADEN  KYLE,  M.D., 
Clinical  Professor  of  Laryngology  and  Rhinology,  Jefferson  Medical 
College,  Philadelphia;  Consulting  Laryngologist,  Rhinologist,  and 
Otologist,  St.  Agnes'  Hospital.  Handsome  octavo  volume  of  about 
630  pages,  with  over  150  illustrations  and  6  lithographic  plates.  Price, 
Cloth,  $ net ;  Half  Morocco,  $ net. 

LAINE'S  TEMPERATURE  CHART. 

Temperature  Chart.  Prepared  by  D.  T.  LAINE,  M.D.  Size  8  x  13^ 
inches.  A  conveniently  arranged  Chart  for  recording  Temperature, 
with  columns  for  daily  amounts  of  Urinary  and  Fecal  Excretions, 
Food,  Remarks,  etc.  On  the  back  of  each  chart  is  given  in  full  the 
method  of  Brand  in  the  treatment  of  Typhoid  Fever.  Price,  per  pad 
of  25  charts,  50  cents  net. 

"  To  the  busy  practitioner  this  chart  will  be  found  of  great  value  in  fever  cases,  and 
especially  for  cases  of  typhoid." — Indian  Lancet,  Calcutta. 

LOCKWOOD'S  PRACTICE  OF  MEDICINE. 

A  Manual  of  the  Practice  of  Medicine.  By  GEORGE  ROE  LOCK- 
WOOD,  M.D.,  Professor  of  Practice  in  the  Woman's  Medical  College 
of  the  New  York  Infirmary,  etc.  935  pages,  with  75  illustrations  in 
the  text,  and  22  full-page  plates.  Cloth,  $2.50  net. 

"  Gives  in  a  most  concise  manner  the  points  essential  to  treatment  usually  enumerated 
in  the  most  elaborate  works." — Massachusetts  Medical  Journal. 

LONG'S  SYLLABUS  OF  QYNECOLOGY. 

A  Syllabus  of  Gynecology,  arranged  in  Conformity  with  «« An 
American  Text-Book  of  Gynecology."  By  J.  W.  LONG,  M.D., 
Professor  of  Diseases  of  Women  and  Children,  Medical  College  of 
Virginia,  etc.  Cloth,  interleaved,  $1.00  net. 

"  The  book  is  certainly  an  admirable  restime  of  what  every  gynecological  student  and 
practitioner  should  know,  and  will  prove  of  value  not  only  to  those  who  have  the  '  American 
Text-Book  of  Gynecology,'  but  to  others  as  well." — Brooklyn  Medical  Journal. 


16  Medical  Publications  of  W.  B.  Saunders. 

MACDONALD'S  SURGICAL  DIAGNOSIS   \ND  TREATMENT. 

Surgical  Diagnosis  and  Treatment.  By  J.  W.  MACDONALD,  M.D. 
Edin.,  F.R.C.S.,  Edin.,  Professor  of  the  Practice  of  Surgery  and  of 
Clinical  Surgery  in  Hamline  University;  Visiting  Surgeon  to  St. 
Barnabas'  Hospital,  Minneapolis,  etc.  Handsome  octavo  volume  of 
800  pages,  profusely  illustrated.  Cloth,  $5.00  net;  Half  Morocco, 
$6.00  net. 

"  A  thorough  and  complete  work  on  surgical  diagnosis  and  treatment,  free  from  pad- 
ding, full  of  valuable  material,  and  in  accord  with  the  surgical  teaching  of  the  day." — The 
Medical  News,  New  York, 

"The  work  is  brimful  of  just  the  kind  of  practical  information  that  is  useful  alike  to 
students  and  practitioners.  It  is  a  pleasure  to  commend  the  book  because  of  its  intrinsic 
value  to  the  medical  practitioner." — Cincinnati  Lancet-Clinic. 

MALLORY  AND  WRIGHT'S  PATHOLOGICAL  TECHNIQUE. 

Pathological  Technique.  A  Practical  Manual  for  Laboratory  Work 
in  Pathology,  Bacteriology,  and  Morbid  Anatomy,  with  chapters  on 
Post- Mortem  Technique  and  the  Performance  of  Autopsies.  By  FRANK 
B.  MALLORY,  A.M.,  M.D.,  Assistant  Professor  of  Pathology,  Harvard 
University  Medical  School,  Boston;  and  JAMES  H.  WRIGHT,  A.M., 
M.D.,  Instructor  in  Pathology,  Harvard  University  Medical  School, 
Boston.  Octavo  volume  of  396  pages,  handsomely  illustrated.  Cloth, 
$2.50  net. 

"  I  have  been  looking  forward  to  the  publication  of  this  book,  and  I  am  glad  to  say  that 
I  find  it  to  be  a  most  useful  laboratory  and  post-mortem  guide,  full  of  practical  information, 
and  well  up  to  date." — WILLIAM  H.  WELCH,  Professor  of  Pathology,  Johns  Hopkins  Uni- 
versity, Baltimore,  Md. 

MARTIN'S   MINOR   SURGERY,   BANDAGING,    AND    VENEREAL 

DISEASES.     Second  Edition,  Revised. 

Essentials  of  Minor  Surgery,  Bandaging,  and  Venereal 
Diseases.  By  EDWARD  MARTIN,  A.M.,  M.D.,  Clinical  Professor  of 
Genito-Urinary  Diseases,  University  of  Pennsylvania,  etc.  Crown 
octavo,  1 66  pages,  with  78  illustrations.  Cloth,  $1.00  ;  interleaved  for 
notes,  $1.25. 

[See  Saunders*  Question- Compends,  page  21.] 

"A  very  practical  and  systematic  study  of  the  subjects,  and  shows  the  author's  famil- 
iarity with  the  needs  of  students." — Therapeutic  Gazette. 

MARTIN'S  SURGERY.     Sixth  Edition,  Revised. 

Essentials  of  Surgery.  Containing  also  Venereal  Diseases,  Surgi- 
cal Landmarks,  Minor  and  Operative  Surgery,  and  a  complete  de- 
scription, with  illustrations,  of  the  Handkerchief  and  Roller  Bandages. 
By  EDWARD  MARTIN,  A.M.,  M.D.,  Clinical  Professor  of  Genito- 
Urinary  Diseases,  University  of  Pennsylvania,  etc.  Crown  octavo,  338 
pages,  illustrated.  With  an  Appendix  containing  full  directions  for  the 
preparation  of  the  materials  used  in  Antiseptic  Surgery,  etc.  Cloth, 
$1.00;  interleaved  for  notes,  $1.25. 

[See  Saunders''  Question- Compends,  page  21.] 

"  Contains  all  necessary  essentials  of  modern  surgery  in  a  comparatively  small  space. 
Its  style  is  interesting,  and  its  illustrations  are  admirable." — Medical  and  Surgical  Reporter. 


Medical  Publications  of  W.  B.  Saunders.  17 

McFARLAND'S   PATHOGENIC   BACTERIA.    Second  Edition,  Re- 
vised and  Greatly  Enlarged. 

Text-Book  upon  the  Pathogenic  Bacteria.  By  JOSEPH  McFAR- 
LAND,  M.  D. ,  Professor  of  Pathology  and  Bacteriology  in  the  Medico- 
Chirurgical  College  of  Philadelphia,  etc.  Octavo  volume  of  497  pages, 
finely  illustrated.  Cloth,  $2.50  net. 

"  Dr.  McFarland  has  treated  the  subject  in  a  systematic  manner,  and  has  succeeded  in 
presenting  in  a  concise  and  readable  form  the  essentials  of  bacteriology  up  to  date.  Alto- 
gether, the  book  is  a  satisfactory  one,  and  I  shall  take  pleasure  in  recommending  it  to  the 
students  of  Trinity  College." — H.  B.  ANDERSON,  M.D.,  Professor  of  Pathology  and  Bac- 
teriology, Trinity  Medical  College,  Toronto. 

MEIGS  ON  FEEDING  IN  INFANCY. 

Feeding  in  Early  Infancy.  By  ARTHUR  V.  MEIGS,  M.D.  Bound 
in  limp  cloth,  flush  edges,  25  cents  net. 

"  This  pamphlet  is  worth  many  times  over  its  price  to  the  physician.  The  author's 
experiments  and  conclusions  are  original,  and  have  been  the  means  of  doing  much  good." — 
Medical  Bulletin. 

MOORE'S  ORTHOPEDIC  SURGERY. 

A  Manual  of  Orthopedic  Surgery.  By  JAMES  E.  MOORE,  M.D., 
Professor  of  Orthopedics  and  Adjunct  Professor  of  Clinical  Surgery, 
University  of  Minnesota,  College  of  Medicine  and  Surgery.  Octavo 
volume  of  356  pages,  handsomely  illustrated.  Cloth,  $2.50  net. 

"  A  most  attractive  work.  The  illustrations  and  the  care  with  which  the  book  is  adapted 
to  the  wants  of  the  general  practitioner  and  the  student  are  worthy  of  great  praise." — Chicago 
Medical  Recorder. 

"  A  very  demonstrative  work,  every  illustration  of  which  conveys  a  lesson.  The  work  is 
a  most  excellent  and  commendable  one,  which  we  can  certainly  endorse  with  pleasure." — 
St.  Louis  Medical  and  Surgical  Journal. 

MORRIS'S   MATERIA    MEDICA   AND  THERAPEUTICS.        Fifth 

Edition,  Revised. 

Essentials  of  Materia  Medica,  Therapeutics,  and  Prescription- 
Writing.  By  HENRY  MORRIS,  M.D.,  late  Demonstrator  of  Thera- 
peutics, Jefferson  Medical  College,  Philadelphia ;  Fellow  of  the  College 
of  Physicians,  Philadelphia,  etc.  Crown  octavo,  288  pages.  Cloth, 
$1.00  j  interleaved  for  notes,  $1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"  This  work,  already  excellent  in  the  old  edition,  has  been  largely  improved  by  revi- 
sion." — American  Practitioner  and  News. 

MORRIS,  WOLFF,  AND  POWELL'S   PRACTICE   OF   MEDICINE. 

Third  Edition,  Revised. 

Essentials  of  the  Practice  of  Medicine.  By  HENRY  MORRIS,  M.D., 
late  Demonstrator  of  Therapeutics,  Jefferson  Medical  College,  Phila- 
delphia ;  with  an  Appendix  on  the  Clinical  and  Microscopic  Examina- 
tion of  Urine,  by  LAWRENCE  WOLFF,  M.  D. ,  Demonstrator  of  Chemistry, 
Jefferson  Medical  College,  Philadelphia.  Enlarged  by  some  300  essen- 
tial formulae  collected  and  arranged  by  WILLIAM  M.  POWELL,  M.D. 
Post-octavo,  488  pages.  Cloth,  $2.00. 

[See  Saunders'  Question- Compends,  page  21.] 

"  The  teaching  is  sound,  the  presentation  graphic  ;  matter  full  as  can  be  desired,  and 
Style  attractive." — American  Practitioner  and  News. 


18  Medical  Publications  of  W.  B.  Saunders. 

MORTEN'S  NURSE'S  DICTIONARY. 

Nurse's  Dictionary  of  Medical  Terms  and  Nursing  Treat- 
ment. Containing  Definitions  of  the  Principal  Medical  and  Nursing 
Terms  and  Abbreviations ;  of  the  Instruments,  Drugs,  Diseases,  Acci- 
dents, Treatments,  Operations,  Foods,  Appliances,  etc.  encountered 
in  the  ward  or  in  the  sick-room.  By  HONNOR  MORTEN,  author  of 
11  How  to  Become  a  Nurse,"  etc.  i6mo,  140  pages.  Cloth,  $1.00. 

*'  A  handy,  compact  little  volume,  containing  a  large  amount  of  general  information,  all 
of  which  is  arranged  in  dictionary  or  encyclopedic  form,  thus  facilitating  quick  reference. 
It  is  certainly  of  value  to  those  for  whose  use  it  is  published." — Chicago  Clinical  Review. 

NANCREDE'S  ANATOMY.     Fifth  Edition. 

Essentials  of  Anatomy,  including  the  Anatomy  of  the  Viscera. 
By  CHARLES  B.  NANCREDE,  M.D.,  Professor  of  Surgery  and  of  Clini- 
cal Surgery  in  the  University  of  Michigan,  Ann  Arbor.  Crown  octavo, 
388  pages;  180  illustrations.  With  an  Appendix  containing  over  60 
illustrations  of  the  osteology  of  the  human  body.  Based  upon  Gray's 
Anatomy.  Cloth,  $1.00;  interleaved  for  notes,  $1.25. 
[See  Saunders*  Question-  Compends,  page  21.] 

"  For  self-quizzing  and  keeping  fresh  in  mind  the  knowledge  of  anatomy  gamed  at 
school,  it  would  not  be  easy  to  speak  of  it  in  terms  too  favorable." — American  Practitioner. 

NANCREDE'S  ANATOMY  AND  DISSECTION.     Fourth  Edition. 
Essentials  of  Anatomy  and    Manual  of   Practical    Dissection. 

By  CHARLES  B.  NANCREDE,  M.D.,  Professor  of  Surgery  and  of  Clinical 
Surgery,  University  of  Michigan,  Ann  Arbor.    Post-octavo ;  500  pages, 
•  with  full-page  lithographic  plates  in  colors,  and  nearly  200  illustrations. 
Extra  Cloth  (or  Oilcloth  for  the  dissection-room),  #2.00  net. 

"  Jt  may  in  many  respects  be  considered  an  epitome  of  Gray's  popular  work  on  general 
anatomy,  at  the  same  time  having  some  distinguishing  characteristics  of  its  own  to  commend 
it.  The  plates  are  of  more  than  ordinary  excellence,  and  are  of  especial  value  to  students 
in  their  work  in  the  dissecting  room." — -Journal  of  the  American  Medical  Association. 

NORRIS'S  SYLLABUS  OF  OBSTETRICS.  Third  Edition,  Revised. 
Syllabus  of  Obstetrical  Lectures  in  the  Medical  Department 
of  the  University  of  Pennsylvania.  By  RICHARD  C.  NORRIS, 
A.M.,  M.D.,  Demonstrator  of  Obstetrics,  University  of  Pennsylvania. 
Crown  octavo,  222  pages.  Cloth,  interleaved  for  notes,  $2.00  net. 

"  This  work  is  so  far  superior  to  others  on  the  same  subject  that  we  take  pleasure  in 
calling  attention  briefly  to  its  excellent  features.  It  covers  the  subject  thoroughly,  and  will 
prove  invaluable  both  to  the  student  and  the  practitioner." — Medical  Record,  New  York. 

PENROSE'S  DISEASES  OF  WOMEN.     Second  Edition,  Revised. 
A  Text-Book  of  Diseases  of  Women.     By  CHARLES  B.  PENROSE, 
M.D.,  PH.D.,  Professor  of  Gynecology  in  the  University  of  Pennsyl- 
vania;   Surgeon    to   the   Gynecean    Hospital,   Philadelphia.     Octavo 
volume  of  529  pages,  handsomely  illustrated.     Cloth,  #3.50  net. 

"I  shall  value  very  highly  the  copy  of  Penrose's  'Diseases  of  Women'  received. 
I  have  already  recommended  it  to  my  class  as  THE  BEST  book."— HOWARD  A.  KELLY, 
Professor  of  Gynecology  and  Obstetrics,  Johns  Hopkins  University,  Baltimore,  Md. 

"  The  book  is  to  be  commended  without  reserve,  not  only  to  the  student  but  to  the 
general  practitioner  who  wishes  to  have  the  latest  and  best  modes  of  treatment  explained 
with  absolute  clearness." — Therapeutic  Gazette. 


Medical  Publications  of  W.  B.  Saunders.  19 

POWELL'S  DISEASES  OF  CHILDREN.     Second  Edition. 

Essentials  of  Diseases  of  Children.  By  WILLIAM  M.  POWELL, 
M.D.,  Attending  Physician  to  the  Mercer  House  for  Invalid  Women 
at  Atlantic  City,  N.  J. ;  late  Physician  to  the  Clinic  for  the  Diseases  of 
Children  in  the  Hospital  of  the  University  of  Pennsylvania.  Crown 
octavo,  222  pages.  Cloth,  $1.00;  interleaved  for  notes,  $1.25. 

[See  Saunders'  Question-  Compends,  page  21.] 

"Contains  the  gist  of  all  the  best  works  in  the  department  to  which  it  relates."— 
American  Practitioner  and  News. 

PRINGLE'S  SKIN  DISEASES  AND  SYPHILITIC  AFFECTIONS. 
Pictorial  Atlas  of  Skin  Diseases  and  Syphilitic  Affections 
(American  Edition).  Translation  from  the  French.  Edited  by 
J.  J.  PRINGLE,  M.B.,  F.R.C.P.,  Assistant  Physician  to  the  Middlesex 
Hospital,  London.  Photo-lithochromes  from  the  famous  models  in 
the  Museum  of  the  Saint-Louis  Hospital,  Paris,  with  explanatory  wood- 
cuts and  text.  In  12  Parts.  Price  per  Part,  $3.00.  Complete  in 
one  volume,  Half  Morocco  binding,  $40.00  net. 

"I  strongly  recommend  this  Atlas.  The  plates  are  exceedingly  well  executed,  and 
will  be  of  great  value  to  all  studying  dermatology." — STEPHEN  MACKENZIE,  M.D. 

' '  The  introduction  of  explanatory  wood-cuts  in  the  text  is  a  novel  and  most  important 
feature  which  greatly  furthers  the  easier  understanding  of  the  excellent  plates,  than  which 
nothing,  we  venture  to  say,  has  been  seen  better  in  point  of  correctness,  beauty,  and  general 
merit." — New  York  Medical  Journal. 

PYE'S  BANDAGING. 

Elementary  Bandaging  and  Surgical  Dressing.  With  Direc- 
tions concerning  the  Immediate  Treatment  of  Cases  of  Emergency. 
For  the  use  of  Dressers  and  Nurses.  By  WALTER  PYE,  F.R.C.S.,  late 
Surgeon  to  St.  Mary's  Hospital,  London.  Small  i2mo,  with  over  80 
illustrations.  Cloth,  flexible  covers,  75  cents  net. 

"  The  directions  are  clear  and  the  illustrations  are  good." — London  Lancet. 
"  The  author  writes  well,  the  diagrams  are  clear,  and  the  book  itself  is  small  and  port- 
able, although  the  paper  and  type  are  good." — British  Medical  Journal. 

RAYMOND'S  PHYSIOLOGY. 

A  Manual  of  Physiology.  By  JOSEPH  H.  RAYMOND,  A.M.,  M.D., 
Professor  of  Physiology  and  Hygiene  and  Lecturer  on  Gynecology  in 
the  Long  Island  College  Hospital ;  Director  of  Physiology  in  the 
Hoagland  Laboratory,  etc.  382  pages,  with  102  illustrations  in  the 
text,  and  4  full-page  colored  plates.  Cloth,  $1.25  net. 

"  Extremely  well  gotten  up,  and  the  illustrations  have  been  selected  with  care.  The 
text  is  fully  abreast  with  modern  physiology." — British  Medical  Journal. 

RONTGEN  RAYS. 

Archives  of  the  Rontgen  Ray  (Formerly  Archives  of  Clinical 
Skiagraphy).  Edited  by  SYDNEY  ROWLAND,  M.A.,  M.R.C.S.,  and 
W.  S.  HEDLEY,  M.D.,  M.R.C.S.  A  series  of  collotype  illustrations, 
with  descriptive  text,  illustrating  the  applications  of  the  new  photo- 
graphy to  Medicine  and  Surgery.  Price  per  Part,  $1.00.  Now  ready: 
Vol.  I  ,  Parts  I.  to  IV.;  Vol.  II.,  Parts  I.,  II. 


Arranged  in  Question  and 
Answer  Form. 

U-CO  1  IwlN  MOST  COMPLETE  AND  BEST 


ILLUSTRATED  SERIES  OF 

COMPENDS  EVER  ISSUED. 

Now  the  Standard  Authorities  in  Medical  Literature 

with  Students  and  Practitioners  in  every  City  of  the  United  States  and  Canada. 


OVER  175,000  COPIES  SOLD, 
THE  REASON  WHY. 

They  are  the  advance  guard  of  "Student's  Helps" — that  DO  HELP.  They  are  the 
leaders  in  their  special  line,  well  and  authoritatively  written  by  able  men,  who,  as  teachers  in 
the  large  colleges,  know  exactly  what  is  wanted  by  a  student  preparing  for  his  examinations. 
The  judgment  exercised  in  the  selection  of  authors  is  fully  demonstrated  by  their  professional 
standing.  Chosen  from  the  ranks  of  Demonstrators,  Quiz-masters,  and  Assistants,  most  of 
them  have  become  Professors  and  Lecturers  in  their  respective  colleges. 

Each  book  is  of  convenient  size  (5x7  inches) ,  containing  on  an  average  250  pages, 
profusely  illustrated,  and  elegantly  printed  in  clear,  readable  type,  on  fine  paper. 

The  entire  series,  numbering  twenty-three  volumes,  has  been  kept  thoroughly  revised 
and  enlarged  when  necessary,  many  of  the  books  being  in  their  fifth  and  sixth  editions. 

TO  SUM  UP. 

Although  there  are  numerous  other  Quizzes,  Manuals,  Aids,  etc.  in  the  market,  none  of 
them  approach  the  "Blue  Series  of  Question  Compends;''  and  the  claim  is  made  for  the 
following  points  of  excellence  : 

1.  Professional  distinction  and  reputation  of  authors. 

2.  Conciseness,  clearness,  and  soundness  of  treatment. 

3.  Quality  of  illustrations,  paper,  printing,  and  binding. 

Any  cf  these  Compends  will  be   mailed  on  receipt  of  price  (see  next  page  for  List). 


Saunders'  Question-Compend  Series* 

Price,  Cloth,  $1.00  per  copy,  except  when  otherwise  noted. 

"  Where  the  work   of  preparing  students'  manuals  is  to  end  we  cannot  say  but  the 
Saunders  Series,  in  our  opinion,  bears  off  the  palm  at  present."— New  York  Medical  Record. 


1.  ESSENTIALS  OF  PHYSIOLOGY.     By  H.  A.  HARE,  M.D.    Fourth  edition, 

revised  and  enlarged.      ($l.oo  net.) 

2.  ESSENTIALS   OF   SURGERY.     By  EDWARD  MARTIN,  M.D.      Sixth  edition, 

revised,  with  an  Appendix  on  Antiseptic  Surgery. 

3.  ESSENTIALS   OF   ANATOMY.      By  CHARLES   B.   NANCREDE,   M.D.     Fifth 

edition,  with  an  Appendix. 

4.  ESSENTIALS  OF  MEDICAL  CHEMISTRY,  ORGANIC  AND  INORGANIC. 

By  LAWRENCE  WOLFF,  M.D.     Fourth  edition,  revised,  with  an  Appendix. 

5.  ESSENTIALS  OF  OBSTETRICS.     By  W.  EASTERLY  ASHTON,  M.D.     Fourth 

edition,  revised  and  enlarged. 

6.  ESSENTIALS  OF  PATHOLOGY  AND  MORBID  ANATOMY.     By  C.  E. 

ARMAND  SEMPLE,  M.D. 

7.  ESSENTIALS  OF  MATERIA  MEDICA,  THERAPEUTICS,  AND  PRE- 

SCRIPTION-WRITING.    By  HENRY  MORRIS,  M.D.       Fifth  edition,  revised. 

8.  9.    ESSENTIALS   OF    PRACTICE   OF   MEDICINE.     By   HENRY  MORRIS, 

M.D.  An  Appendix  on  URINE  EXAMINATION.  By  LAWRENCE  WOLFF,  M.D. 
Third  edition,  enlarged  by  some  300  Essential  Formulae,  selected  from  eminent 
authorities,  by  WM.  M.  POWELL,  M.D.  (Double  number,  $2.00.) 

10.  ESSENTIALS  OF  GYN/CCOLOGY.      By  EDWIN  B.  CRAGIN,  M.D.      Fourth 

edition,  revised. 

11.  ESSENTIALS  OF  DISEASES  OF  THE  SKIN.     By  HENRY  W.  STELWAGON, 

M.D.     Third  edition,  revised  and  enlarged.     ($l.oo  net.) 

12.  ESSENTIALS  OF  MINOR  SURGERY,  BANDAGING,  AND  VENEREAL 

DISEASES.     By  EDWARD  MARTIN,  M.D.     Second  ed. ,  revised  and  enlarged. 

13.  ESSENTIALS  OF  LEGAL  MEDICINE,  TOXICOLOGY,  AND  HYGIENE. 

By  C.  E.  ARMAND  SEMPLE,  M.D. 

14.  ESSENTIALS  OF   DISEASES  OF  THE   EYE,  NOSE,  AND  THROAT. 

By  EDWARD  JACKSON,  M.D.,  and  E.  B.  GLEASON,  M.D.     Second  ed.,  revised. 

15.  ESSENTIALS  OF  DISEASES  OF  CHILDREN.     By  WILLIAM  M.  POWELL, 

M.D.     Second  edition. 

16.  ESSENTIALS  OF   EXAMINATION   OF   URINE.     By  LAWRENCE  WOLFF, 

M.D.     Colored  "  VOGEL  SCALE."     (75  cents.) 

17.  ESSENTIALS  OF  DIAGNOSIS.     By  S.  SOLIS  COHEN,  M.D.,and  A.  A.  ESHNER, 

M.D.      ($1.50  net.) 

18.  ESSENTIALS  OF  PRACTICE   OF   PHARMACY.     By  Lucius   E.    SAYRE. 

Second  edition,  revised  and  enlarged. 

20.  ESSENTIALS  OF  BACTERIOLOGY.     By  M.  V.  BALL,  M.D.     Third  edition, 

revised. 

21.  ESSENTIALS  OF  NERVOUS  DISEASES  AND  INSANITY.     By  JOHN  C. 

SHAW,  M.D.     Third  edition,  revised. 

22.  ESSENTIALS  OF   MEDICAL  PHYSICS.      By  FRED  J.    BROCKWAY,   M.D. 

Second  edition,  revised.      ($I.OO  net.) 
.23.    ESSENTIALS  OF  MEDICAL  ELECTRICITY.    By  DAVID  D.  STEWART,  M.D., 

and  EDWARD  S.  LAWRANCE,  M.D. 
24.    ESSENTIALS  OF  DISEASES  OF  THE   EAR.      By  E.  B.  GLEASON,  M.D. 

Second  edition,  revised  and  greatly  enlarged. 


Pamphlet  containing  specimen  pages,  etc.  sent  free  upon  application* 


Saunders' 
New  Series 
of  Manuals 


for  Students 
and 
Practitioners, 


'TrHAT  there  exists  a  need  for  thoroughly  reliable  hand-books  on  the  leading  branches 
of  Medicine  and  Surgery  is  a  fact  amply  demonstrated  by  the  favor  with  which 
the  SAUNDERS  NEW  SERIES  OF  MANUALS  have  been  received  by  medical 
students  and  practitioners  and  by  the  Medical  Press.  These  manuals  are  not  merely 
condensations  from  present  literature,  but  are  ably  written  by  well-known  authors 
and  practitioners,  most  of  them  being  teachers  in  representative  American  colleges. 
Each  volume  is  concisely  and  authoritatively  written  and  exhaustive  in  detail,  without 
being  encumbered  with  the  introduction  of  "cases,"  which  so  largely  expand  the 
ordinary  text-book.  These  manuals  will  therefore  form  an  admirable  collection  of 
advanced  lectures,  useful  alike  to  the  medical  student  and  the  practitioner:  to  the 
latter,  too  busy  to  search  through  page  after  page  of  elaborate  treatises  for  what  he 
wants  to  know,  they  will  prove  of  inestimable  value ;  to  the  former  they  will  afford 
safe  guides  to  the  essential  points  of  study. 

The  SAUNDERS  NEW  SERIES  OF  MANUALS  are  conceded  to  be  superior 
to  any  similar  books  now  on  the  market.  No  other  manuals  afford  so  much  infor- 
mation in  such  a  concise  and  available  form.  A  liberal  expenditure  has  enabled  the 
publisher  to  render  the  mechanical  portion  of  the  work  -worthy  of  the  high  literary 
standard  attained  by  these  books. 

Any  of  these  Manuals  will  be  mailed  on  receipt  of  price  (see  next  page  for  List). 


Saunders'  New  Series  of  Manuals. 


VOLUMES   PUBLISHED. 

PHYSIOLOGY.  By  JOSEPH  HOWARD  RAYMOND,  A.M.,  M.D.,  Professor  of  Physiology 
and  Hygiene  and  Lecturer  on  Gynecology  in  the  Long  Island  College  Hospital; 
Director  of  Physiology  in  the  Hoagland  Laboratory,  etc.  Illustrated.  Cloth,  #1.25  net. 

SURGERY,  General  and  Operative.  By  JOHN  CHALMERS  DACOSTA,  M.D.,  Clini- 
cal Professor  of  Surgery,  Jefferson  Medical  College,  Philadelphia;  Surgeon  to  the 
Philadelphia  Hospital,  etc.  Second  edition,  thoroughly  revised  and  greatly  enlarged. 
Octavo,  911  pages,  profusely  illustrated.  Cloth,  $4.00  net ;  Half  Morocco,  $5.00  net. 

DOSE-BOOK    AND    MANUAL    OF    PRESCRIPTION-WRITING.     By   E.    Q. 

THORNTON,  M.D.,  Demonstrator  of  Therapeutics,  Jefferson  Medical  College,  Phila-' 
delphia.     Illustrated.     Cloth,  $1.25  net. 

SURGICAL  ASEPSIS.  By  CARL  BECK,  M.D.,  Surgeon  to  St.  Mark's  Hospital  and 
to  the  New  York  German  Poliklinik,  etc.  Illustrated.  Cloth,  $1.25  net. 

MEDICAL  JURISPRUDENCE.  By  HENRY  C.  CHAPMAN,  M.D.  Professor  of  Insti- 
tutes of  Medicine  and  Medical  Jurisprudence  in  the  Jefferson  Medical  College  of  Phila- 
delphia. Illustrated.  Cloth,  $1.50  net. 

SYPHILIS  AND  THE  VENEREAL  DISEASES.  By  JAMES  NEVINS  HYDE,  M.D., 
Professor  of  Skin  and  Venereal  Diseases,  and  FRANK  H.  MONTGOMERY,  M.D., 
Lecturer  on  Dermatology  and  Genito-Urinary  Diseases  in  Rush  Medical  College, 
Chicago.  Profusely  illustrated.  Cloth,  $2.50  net. 

PRACTICE  OF  MEDICINE.  By  GEORGE  ROE  LOCKWOOD,  M.D.,  Professor  of 
Practice  in  the  Woman's  Medical  College  of  the  New  York  Infirmary ;  Instructor  in 
Physical  Diagnosis  in  the  Medical  Department  of  Columbia  College,  etc.  Illustrated. 
Cloth,  $2.50  net. 

MANUAL  OF  ANATOMY.  By  IRVING  S.  HAYNES,  M.D.,  Adjunct  Professor  of 
Anatomy  and  Demonstrator  of  Anatomy,  Medical  Department  of  the  New  York 
University,  etc.  Beautifully  illustrated.  Cloth,  $2.50  net. 

MANUAL  OF  OBSTETRICS.  By  W.  A.  NEWMAN  DORLAND,  M.D.,  Assistant 
Demonstrator  of  Obstetrics,  University  of  Pennsylvania ;  Chief  of  Gynecological  Dis- 
pensary, Pennsylvania  Hospital,  etc.  Profusely  illustrated.  Cloth,  $2.50  net. 

DISEASES  OF  WOMEN.  By  J.  BLAND  SUTTON,  F.  R.  C.  S.,  Assistant  Surgeon  to 
Middlesex  Hospital  and  Surgeon  to  Chelsea  Hospital,  London;  and  ARTHUR  E. 
GILES,  M.  D.,  B.  Sc.  Lond.,  F.R.C.S.  Edin.,  Assistant  Surgeon  to  Chelsea  Hospital, 
London.  Handsomely  illustrated.  Cloth,  $2.50  net.  ^ 


VOLUMES  IN  PREPARATION. 

NOSE  AND  THROAT.  By  D.  BRADEN  KYLE,  M.D.,  Clinical  Professor  of  Laryn- 
gology and  Rhinology,  Jefferson  Medical  College,  Philadelphia  ;  Consulting  Laryngolo- 
gist,  Rhinologist,  and  Otologist,  St.  Agnes'  Hospital ;  Bacteriologist  to  the  Philadel- 
phia Orthopedic  Hospital  and  Infirmary  for  Nervous  Diseases,  etc. 

NERVOUS  DISEASES.  By  CHARLES  W.  BURR,  M.D.,  Clinical  Professor  of  Nervous 
Diseases,  Medico-Chirurgical  College.  Philadelphia;  Pathologist  to  the  Orthopaedic 
Hospital  and  Infirmary  for  Nervous  Diseases;  Visiting  Physician  to  the  St.  Joseph 
Hospital,  etc. 

V  There  will  be  published  in  the  same  series,  at  short  intervals,  carefully-prepared  works 
on  various  subjects  by  prominent  specialists. 


Pamphlet  containing  specimen  pages,  etc.  sent  free  upon  application. 


24  Medical  Publications  of  W.  B.  Saunders. 

SAUNDBY'S  RENAL  AND  URINARY  DISEASES. 

Lectures  on  Renal  and  Urinary  Diseases.  By  ROBERT  SAUNDBY, 
M.D.  Edin.,  Fellow  of  the  Royal  College  of  Physicians,  London,  and 
of  the  Royal  Medico-Chirurgical  Society ;  Physician  to  the  General 
Hospital ;  Consulting  Physician  to  the  Eye  Hospital  and  to  the  Hos- 
pital for  Diseases  of  Women;  Professor  of  Medicine  in  Mason  College, 
Birmingham,  etc.  Octavo  volume  of  434  pages,  with  numerous  illus- 
trations and  4  colored  plates.  Cloth,  $2.50  net. 

"  The  volume  makes  a  favorable  impression  at  once.  The  style  is  clear  and  succinct. 
We  cannot  find  any  part  of  the  subject  in  which  the  views  expressed  are  not  carefully  thought 
out  and  fortified  by  evidence  drawn  from  the  most  recent  sources.  The  book  may  be  cordially 
recommended.' ' — British  Medical  Journal. 

SAUNDERS'  MEDICAL  HAND-ATLASES. 

This  series  of  books  consists  of  authorized  translations  into  English  of 
the  world-famous  Lehmann  Medicinische  Handatlanten.  Each 
volume  contains  from  50  to  100  colored  lithographic  plates,  besides 
numerous  illustrations  in  the  text.  There  is  a  full  description  of  each 
plate,  and  each  book  contains  a  condensed  but  adequate  outline  of  the 
subject  to  which  it  is  devoted.  For  full  description  of  this  series,  with 
list  of  volumes  and  prices,  see  page  2. 

"  Lehmann  Medicinische  Handatlanten  belong  to  that  class  of  books  that  are  too  good 
to  be  appropriated  by  any  one  nation." — Journal  of  Eye,  Ear,  and  Throat  Diseases. 

"  The  appearance  of  these  works  marks  a  new  era  in  illustrated  English  medical 
works." — The  Canadian  Practitioner. 

SAUNDERS'   POCKET  MEDICAL   FORMULARY.      Fifth  Edition, 
Revised. 

By  WILLIAM  M.  POWELL,  M.D.,  Attending  Physician  to  the  Mercer 
House  for  Invalid  Women  at  Atlantic  City,  N.  J.  Containing  1800 
formulae  selected  from  the  best-known  authorities.  With  an  Appen- 
dix containing  Posological  Table,  Formulae  and  Doses  for  Hypo- 
dermic Medication,  Poisons  and  their  Antidotes,  Diameters  of  the 
Female  Pelvis  and  Foetal  Head,  Obstetrical  Table,  Diet  List  for  Various 
Diseases,  Materials  and  Drugs  used  in  Antiseptic  Surgery,  Treatment 
of  Asphyxia  from  Drowning,  Surgical  Remembrancer,  Tables  of 
Incompatibles,  Eruptive  Fevers,  Weights  and  Measures,  etc.  Hand- 
somely bound  in  flexible  morocco,  with  side  index,  wallet,  and  flap. 
|i!75  net. 

"This  little  book,  that  can  be  conveniently  carried  in  the  pocket,  contains  an  immense 
amount  of  material.  It  is  very  useful,  and,  as  the  name  of  the  author  of  each  prescription 
is  given,  is  unusually  reliable." — Medical  Record,  New  York. 

SAYRE'S  PHARMACY.     Second  Edition,  Revised. 

Essentials  of  the  Practice  of  Pharmacy.  By  Lucius  E.  SAYRE, 
M.D.,  Professor  of  Pharmacy  and  Materia  Medica  in  the  University  of 
Kansas.  Crown  octavo,  200  pages.  Cloth,  $1.00;  interleaved  for 
notes,  $1.25. 

[See  Sounders'  Question- Compends,  page  21.] 

"  The  topics  are  treated  in  a  simple,  practical  manner,  and  the  work  forms  a  very  useful 
student's  manual." — Boston  Medical  and  Surgical  Journal. 


Medical  Publications  of  W.  B.  Saunders.  25 

SEMPLE'S  LEGAL  MEDICINE,  TOXICOLOGY,  AND  HYGIENE. 
Essentials  of   Legal   Medicine,  Toxicology,  and  Hygiene.     By 

C.  E.  ARMAND  SEMPLE,  B.  A.,  M.  B.  Cantab.,  M.  R.  C.  P.  Lond., 
Physician  to  the  Northeastern  Hospital  for  Children,  Hackney,  etc. 
Crown  octavo,  2 1 2  pages ;  130  illustrations.  Cloth,  $1.00;  interleaved 
for  notes,  $1.25. 

[See  Sounders'  Question- Compends,  page  21.] 

"  No  general  practitioner  or  student  can  afford  to  be  without  this  valuable  work.  The 
subjects  are  dealt  with  by  a  masterly  hand." — London  Hospital  Gazette. 

SEMPLE'S  PATHOLOGY  AND  MORBID  ANATOMY. 

Essentials    of    Pathology    and    Morbid    Anatomy.      By  C.   E. 

ARMAND  SEMPLE,  B.A.,  M.B.  Cantab.,  M.R.C.P.  Lond.,  Physician  to 
the  Northeastern  Hospital  for  Children,  Hackney,  etc.    Crown  octavo, 
1 74  pages;  illustrated.      Cloth,  $1.00;  interleaved  for  notes,  $1.25. 
[See  Saunders'  Question- Compends,  page  21.] 

"  Should  take  its  place  among  the  standard  volumes  on  the  bookshelf  of  both  student 
and  practitioner." — London  Hospital  Gazette. 

SENN'S  GENITO-URINARY  TUBERCULOSIS. 

Tuberculosis  of  the  Genito-Urinary  Organs,  Male  and  Female. 

By  NICHOLAS  SENN,  M.D.,  PH.D.,  LL.D.,  Professor  of  the  Practice  of 
Surgery  and  of  Clinical  Surgery,  Rush  Medical  College,  Chicago. 
Handsome  octavo  volume  of  320  pages,  illustrated.  Cloth,  $3.00  net. 

"  An  important  book  upon  an  important  subject,  and  written  by  a  man  of  mature  judg- 
ment and  wide  experience.  The  author  has  given  us  an  instructive  book  upon  one  of  the 
most  important  subjects  of  the  day." — Clinical  Reporter. 

"  A  work  which  adds  another  to  the  many  obligations  the  profession  owes  the  talented 
author." — Chicago  Medical  Recorder. 

SENN'S  SYLLABUS  OF  SURGERY. 

A  Syllabus  of  Lectures  on  the  Practice  of  Surgery,  arranged 
in  conformity  with  «« An  American  Text-Book  of  Surgery."  By 

NICHOLAS  SENN,  M.D.,  PH.D.,  Professor  of  the  Practice  of  Surgery  and 
of  Clinical  Surgery  in  Rush  Medical  College,  Chicago.  Cloth,  $2.00. 

"  This  syllabus  will  be  found  of  service  by  the  teacher  as  well  as  the  student,  the  work 
being  superbly  done.  There  is  no  praise  too  high  for  it.  No  surgeon  should  be  without 
it. " — New  York  Medical  Times. 

SENN'S  TUMORS. 

Pathology  and  Surgical  Treatment  of  Tumors.     By  N.  SENN, 

M.D.,  PH.D.,  LL.D.,  Professor  of  Surgery  and  of  Clinical  Surgery, 
Rush  Medical  College ;  Professor  of  Surgery,  Chicago  Polyclinic ; 
Attending  Surgeon  to  Presbyterian  Hospital;  Surgeon-in-Chief,  St. 
Joseph's  Hospital,  Chicago.  Octavo  volume  of  710  pages,  with  515 
engravings,  including  full-page  colored  plates.  Cloth,  $6.00  net; 
Half  Morocco,  $7.00  net. 

"  The  most  exhaustive  of  any  recent  book  in  English  on  this  subject.  It  is  well  illus- 
trated, and  will  doubtless  remain  as  the  principal  monograph  on  the  subject  in  our  language 
for  some  years.  The  book  is  handsomely  illustrated  and  printed,  and  the  author  has  given  a 
notable  and  lasting  contribution  to  surgery." — Journal  of  the  American  Medical  Association. 


26  Medical  Publications  of  W.  B.  Saunders. 

SHAW'S  NERVOUS  DISEASES  AND  INSANITY.     Third  Edition, 

Revised. 

Essentials  of  Nervous  Diseases  and  Insanity.  By  JOHN  C. 
SHAW,  M.D.,  Clinical  Professor  of  Diseases  of  the  Mind  and  Nervous 
System,  Long  Island  College  Hospital  Medical  School ;  Consulting 
Neurologist  to  St.  Catherine's  Hospital  and  to  the  Long  Island  College 
Hospital.  Crown  octavo,  186  pages;  48  original  illustrations.  Cloth, 
$1.00  ;  interleaved  for  notes,  $1.25. 

[See  Saunders1  Question-  Compends,  page  21.] 

"Clearly  and  intelligently  written." — Boston  Medical  and  Surgical  Journal. 

"There  is  a  mass  of  valuable  material  crowded  into  this  small  compass." — American 
Medico- Surgical  Bulletin. 

STARR'S  DIETS  FOR  INFANTS  AND  CHILDREN. 

Diets  for  Infants  and  Children  in  Health  and  in  Disease.     By 

Louis  STARR,  M.D.,  Editor  of  "An  American  Text-Book  of  the 
Diseases  of  Children."  230  blanks  (pocket-book  size),  perforated 
and  neatly  bound  in  flexible  morocco.  $1.25  net. 

The  first  series  of  blanks  are  prepared  for  the  first  seven  months  of  infant  life  ;  each 
blank  indicates  the  ingredients,  but  not  the  quantities,  of  the  food,  the  latter  directions  being 
left  for  the  physician.  After  the  seventh  month,  modifications  being  less  necessary,  the  diet 
lists  are  printed  in  full.  Formulae  for  the  preparation  of  diluents  and  foods  are  appended. 

STELW AGON'S  DISEASES  OF  THE  SKIN.  Third  Edition,  Revised. 
Essentials  of  Diseases  of  the  Skin.  By  HENRY  W.  STELWAGON, 
M.D.,  Clinical  Professor  of  Dermatology  in  the  Jefferson  Medical 
College,  Philadelphia ;  Dermatologist  to  the  Philadelphia  Hospital ; 
Physician  to  the  Skin  Department  of  the  Howard  Hospital,  etc. 
Crown  octavo,  270  pages;  86  illustrations.  Cloth,  $i.oonet;  inter- 
leaved for  notes,  $1.25  net. 

[See  Saunders'  Question- Compends,  page  21.] 
"  The  best  student's  manual  on  skin  diseases  we  have  yet  seen." — Times  and  Register. 

STENGEL'S  PATHOLOGY.      Second  Edition. 

A  Text-Book  of  Pathology.  By  ALFRED  STENGEL,  M.  D.,  Physician 
to  the  Philadelphia  Hospital ;  Clinical  Professor  of  Medicine  in  the 
Woman's  Medical  College  ;  Physician  to  the  Children's  Hospital ; 
late  Pathologist  to  the  German  Hospital,  Philadelphia,  etc.  Handsome 
octavo  volume  of  848  pages,  with  nearly  400  illustrations,  many  of  them 
in  colors.  Cloth,  $4.00  net;  Half  Morocco,  $5.00  net. 

STEVENS'  MATERIA   MEDICA   AND   THERAPEUTICS.      Second 

Edition,  Revised. 
A  Manual  of   Materia   Medica   and  Therapeutics.      By  A.   A. 

STEVENS,  A.M.,  M.D.,  Lecturer  on  Terminology  and  Instructor  in 
I'hvMrul  Diagnosis  in  the  University  of  Pennsylvania;  Professor  of 
Pathology  in  the  Woman's  Medical  College  of  Pennsylvania.  Post- 
octavo,  445  pages.  Flexible  leather,  $2.25. 

"The  author  has  faithfully  presented  modern  therapeutics  in  a  comprehensive  work, 
and,  while  intended  particularly  for  the  use  of  students,  it  will  be  found  a  reliable  guide  and 
sufficiently  comprehensive  for  the  physician  in  practice." — University  Medical  Magazine. 


Medical  Publications  of  W.  B.  Saunders.  27 

STEVENS'  PRACTICE  OF  MEDICINE.     Fifth  Edition,  Revised. 
A  Manual  of  the  Practice  of  Medicine.     By  A.  A.  STEVENS,  A.  M., 

M.  D.,  Lecturer  on  Terminology  and  Instructor  in  Physical  Diagnosis 
in  the    University  of   Pennsylvania;    Professor   of   Pathology  in   the 
Woman's   Medical  College  of  Pennsylvania.     Specially  intended   for 
students   preparing  for   graduation  and   hospital  examinations.     Post- 
octavo,  519  pages;   illustrated.     Flexible  leather,  $2.00  net. 
"  The  frequency  with  which  new  editions  of  this  manual  are  demanded  bespeaks  its 
popularity.     It  is  an  excellent  condensation  of  the  essentials  of  medical  practice  for  the 

student,  and  maybe  found  also  an  excellent  reminder  for  the  busy  physician." Buffalo 

Medical  Journal. 

STEWART'S  PHYSIOLOGY.      Third  Edition,  Revised. 

A  Manual  of  Physiology,  with  Practical  Exercises.  For 
Students  and  Practitioners.  By  G.  N.  STEWART,  M.A.,  M.D., 
D.Sc.,  lately  Examiner  in  Physiology,  University  of  Aberdeen,  and 
of  the  New  Museums,  Cambridge  University ;  Professor  of  Physiology 
in  the  Western  Reserve  University,  Cleveland,  Ohio.  Octavo  volume 
of  848  pages;  300  illustrations  in  the  text,  and  5  colored  plates. 
Cloth,  $3.75  net. 

"  It  will  make  its  way  by  sheer  force  of  merit,  and  amply  deserves  to  do  so.  It  is  one 
of  the  very  best  English  text-books  on  the  subject." — London  Lancet.  < 

"Of  the  many  text-books  of  physiology  published,  we  do  not  know  of  one  that  so 
nearly  comes  up  to  the  ideal  as  does  Prof.  Stewart's  volume." — British  Medical  Journal. 

STEWART  AND  LAWRANCE'S  MEDICAL  ELECTRICITY. 

Essentials  of  Medical  Electricity.  By  D.  D.  STEWART,  M.D., 
Demonstrator  of  Diseases  of  the  Nervous  System  and  Chief  of  the 
Neurological  Clinic  in  the  Jefferson  Medical  College;  and  E.  S. 
LAWRANCE,  M.D.,  Chief  of  the  Electrical  Clinic  and  Assistant  Demon- 
strator of  Diseases  of  the  Nervous  System  in  the  Jefferson  Medical 
College,  etc.  Crown  octavo,  158  pages;  65  illustrations.  Cloth, 
$1.00  ;  interleaved  for  notes,  $1.25. 

[See  Saunders'  Question-  Compends,  page  21.] 

"  Throughout  the  whole  brief  space  at  their  command  the  authors  show  a  discriminating 
knowledge  of  their  subject." — Medical  News. 

STONEY'S  NURSING.     Second  Edition,  Revised. 

Practical  Points  in  Nursing.     For  Nurses  in  Private  Practice. 

By  EMILY  A.  M.  STONEY,  Graduate  of  the  Training-School  for  Nurses, 
Lawrence,  Mass.;  late  Superintendent  of  the  Training-School  for 
Nurses,  Carney  Hospital,  South  Boston,  Mass.  456  pages,  illustrated 
with  73  engravings  in  the  text,  and  8  colored  and  half-tone  plates. 
Cloth,  $1.75  net. 

"  There  are  few  books  intended  for  non -professional  readers  which  can  be  so  cordially 
endorsed  by  a  medical  journal  as  can  this  one." — Therapeutic  Gazette. 

"  This  is  a  well- written,  eminently  practical  volume,  which  covers  the  entire  range  of 
private  nursing  as  distinguished  from  hospital  nursing,  and  instructs  the  nurse  how  best  to 
meet  the  various  emergencies  which  may  arise,  and  how  to  prepare  everything  ordinarily 
needed  in  the  illness  of  her  patient." — American  Journal  of  Obstetrics  and  Diseases  of 
Women  and  Children. 

"  It  is  a  work  that  the  physician  can  place  in  the  hands  of  his  private  nurses  with  the 
assurance  of  benefit." — Ohio  Medical  Journal. 


28  Medical  Publications  of  W.  B.  Saunders. 

STONEY'S   MATERIA   MEDICA    FOR   NURSES. 

Materia  Medica  for  Nurses.  By  EMILY  A.  M.  STONEY,  Graduate  of 
the  Training-School  for  Nurses,  Lawrence,  Mass.  ;  late  Superintendent 
of  the  Training-School  for  Nurses,  Carney  Hospital,  South  Boston,  Mass. 
Handsome  octavo  volume  of  306  pages.  Cloth,  $1.50  net. 

The  present  book  differs  from  other  similar  works  in  several  features,  all  of  which  are 
intended  to  render  it  more  practical  and  generally  useful.  The  general  plan  of  the  contents 
follows  the  lines  laid  down  in  training-schools  for  nurses,  but  the  book  contains  much  use- 
ful matter  not  usually  included  in  works  of  this  character,  such  as  Poison-emergencies, 
Ready  Dose-list,  Weights  and  Measures,  etc.,  as  well  as  a  Glossary,  defining  all  the  terms 
used  in  Materia  Medica,  and  describing  all  the  latest  drugs  and  remedies,  which  have  been 
generally  neglected  by  other  books  of  the  kind. 

SUTTON  AND  GILES'  DISEASES  OF  WOMEN. 

Diseases  of  Women.  By  J.  BLAND  SUTTON,  F.R.C.S.,  Assistant 
Surgeon  to  Middlesex  Hospital,  and  Surgeon  to  Chelsea  Hospital, 
London;  and  ARTHUR  E.  GILES,  M.D.,  B.Sc.  Lond. ,  F.R. C.S.  Edin., 
Assistant  Surgeon  to  Chelsea  Hospital,  London.  436  pages,  hand- 
somely illustrated.  Cloth,  $2.50  net. 

"The  text  has  been  carefully  prepared.  Nothing  essential  has  been  omitted,  and  its 
teachings  are  those  recommended  by  the  leading  authorities  of  the  day." — Journal  of  the 
American  Medical  Association. 

THOMAS'S  DIET  LISTS  AND  SICK-ROOM  DIETARY. 

Diet  Lists  and  Sick-Room  Dietary.  By  JEROME  B.  THOMAS, 
M.D.,  Visiting  Physician  to  the  Home  for  Friendless  Women  and 
Children  and  to  the  Newsboys'  Home  ;  Assistant  Visiting  Physician 
to  the  Kings  County  Hospital.  Cloth,  $1.50.  Send  for  sample  sheet. 

THORNTON'S  DOSE-BOOK  AND  PRESCRIPTION-WRITING. 

Dose-Book  and  Manual  of   Prescription-Writing.      By   E.    Q. 

THORNTON,  M.D.,  Demonstrator  of  Therapeutics,  Jefferson  Medical 
College,  Philadelphia.  334  pages,  illustrated.  Cloth,  $1.25  net. 

"Full  of  practical  suggestions;  will  take  its  place  in  the  front  rank  of  works  of  this 
sort." — Medical  Record,  New  York. 

VAN  VALZAH  AND  NISBET'S  DISEASES  OF  THE  STOMACH. 
Diseases  of  the  Stomach.  By  WILLIAM  W.  VAN  VALZAH,  M.D., 
Professor  of  General  Medicine  and  Diseases  of  the  Digestive  System 
and  the  Blood,  New  York  Polyclinic ;  and  J.  DOUGLAS  NISBET,  M.D., 
Adjunct  Professor  of  General  Medicine  and  Diseases  of  the  Digestive 
System  and  the  Blood,  New  York  Polyclinic.  Octavo  volume  of  674 
pages,  illustrated.  Cloth,  $3.50  net. 

"  Its  chief  claim  lies  in  its  clearness  and  general  adaptability  to  the  practical  needs  of 
the  general  practitioner  or  student.  In  these  relations  it  is  probably  the  bevst  of  the  recent 
special  works  on  diseases  of  the  stomach." — Chicago  Clinical  Review. 

VECKI'S   SEXUAL  IMPOTENCE. 

The  Pathology  and  Treatment  of  Sexual  Impotence.  By  VICTOR 
(I.  VECKI,  .M.I).  From  the  second  German  edition,  revised  and  en- 
larged. Demi-octavo,  about  300  pages.  Cloth,  $2.00  net. 

The  subject  of  impotence  has  seldom  been  treated  in  this  country  in  the  truly  scientific 
spirit  that  it  deserves.  Dr.  Vecki's  work  has  long  been  favorably  known,  and  the  German 
book  has  received  the  highest  consideration.  This  edition  is  more  than  a  mere  translation, 
for,  although  based  on  the  German  edition,  it  has  been  entirely  rewritten  in  English. 


Medical  Publications  of  W.  B.  Saunders.  29 

VIERORDT'S  MEDICAL  DIAGNOSIS.  Fourth  Edition,  Revised. 
Medical  Diagnosis.  By  Dr.  OSWALD  VIERORDT,  Professor  of  Medi- 
cine at  the  University  of  Heidelberg.  Translated,  with  additions, 
from  the  fifth  enlarged  German  edition,  with  the  author's  permission, 
by  FRANCIS  H.  STUART,  A.  M.,  M.  D.  Handsome  royal  octavo  volume 
of  603  pages;  194  fine  wood-cuts  in  text,  many  of  them  in  colors. 
Cloth,  $4.00  net;  Sheep  or  Half  Morocco,  $5.00  net. 

"  A  treasury  of  practical  information  which  will  be  found  of  daily  use  to  every  busy 
practitioner  who  will  consult  it." — C.  A.  LINDSLEY,  M.D.,  Professor  of  the  Theory  and 
Practice  of  Medicine,  Yale  University. 

"  Rarely  is  a  b«ok  published  with  which  a  reviewer  can  find  so  little  fault  as  with  the 
volume  before  us.  Each  particular  item  in  the  consideration  of  an  organ  or  apparatus,  which 
is  necessary  to  determine  a  diagnosis  of  any  disease  of  that  organ,  is  mentioned ;  nothing 
seems  forgotten.  The  chapters  on  diseases  of  the  circulatory  and  digestive  apparatus  and 
nervous  system  are  especially  full  and  valuable.  The  reviewer  would  repeat  that  the  book  is 
one  of  the  best — probably  the  best — wjiich  has  fallen  into  his  hands." — University  Medical 
Magazine. 

WARREN'S  SURGICAL  PATHOLOGY  AND  THERAPEUTICS. 

Surgical  Pathology  and  Therapeutics.  By  JOHN  COLLINS  WARREN, 
M.D.,  LL.D.,  Professor  of  Surgery,  Medical  Department  Harvard 
University;  Surgeon  to  the  Massachusetts  General  Hospital,  etc. 
Handsome  octavo  volume  of  832  pages;  136  relief  and  lithographic 
illustrations,  33  of  which  are  printed  in  colors,  and  all  of  which  were 
drawn  by  William  J.  Kaula  from  original  specimens.  Cloth,  $6.00 
net;  Half  Morocco,  $7.00  net. 

"There  is  the  work  of  Dr.  Warren,  which  I  think  is  the  most  creditable  book  on 
Surgical  Pathology,  and  the  most  beautiful  medical  illustration  of  the  bookmaker's  art,  that 
has  ever  been  issued  from  the  American  press." — DR.  ROSWELL  PARK,  in  the  Harvard 
Graduate  Magazine. 

"  The  handsomest  specimen  of  bookmaking  that  has  ever  been  issued  from  the  American 
medical  press." — American  Journal  of  the  Medical  Sciences. 

"  A  most  striking  and  very  excellent  feature  of  this  book  is  its  illustrations.  Without 
exception,  from  the  point  of  accuracy  and  artistic  merit,  they  are  the  best  ever  seen  in  a  work 
of  this  kind.  Many  of  those  representing  microscopic  pictures  are  so  perfect  in  their  coloring 
and  detail  as  almost  to  give  the  beholder  the  impression  that  he  is  looking  down  the  barrel 
of  a  microscope  at  a  well-mounted  section." — Annals  of  Surgery. 

WOLFF  ON  EXAMINATION  OF  URINE. 

Essentials  of  Examination  of  Urine.  By  LAWRENCE  WOLFF,  M.D., 
Demonstrator  of  Chemistry,  Jefferson  Medical  College,  Philadelphia, 
etc.  Colored  (Vogel)  urine  scale  and  numerous  illustrations.  Crown 
octavo.  Cloth,  75  cents. 

[See  Saunders'  Question-  Compends,  page  21.] 
"  A  very  good  work  of  its  kind — very  well  suited  to  its  purpose." — Times  and  Register. 

WOLFF'S  MEDICAL  CHEMISTRY.     Fourth  Edition,  Revised. 
Essentials    of    Medical    Chemistry,   Organic    and    Inorganic. 

Containing  also  Questions  on  Medical  Physics,  Chemical  Physiology, 
Analytical  Processes,  Urinalysis,  and  Toxicology.  By  LAWRENCE 
WOLFF,  M.D.,  Demonstrator  of  Chemistry,  Jefferson  Medical  College, 
Philadelphia,  etc.  Crown  octavo,  218  pages.  Cloth,  $1.00;  inter- 
leaved for  notes,  $1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"The  scope  of  this  work  is  certainly  equal  to  that  of  the  best  course  of  lectures  on 
Medical  Chemistry." — Pharmaceutical  Era. 


CLASSIFIED    LIST 


MEDICAL  PUBLICATIONS 


OF 


W.  B,  SAUNDERS, 

925  Walnut  Street,  Philadelphia. 


ANATOMY,  EMBRYOLOGY, 
HISTOLOGY. 

Clarkson— A  Text-Book  of  Histology,  9 
Haynes — A  Manual  of  Anatomy, ...  13 
Heisler— A  Text- Book  of  Embryology,  13 
Nancrede — Essentials  of  Anatomy,  .  .  18 
Nancrede — Essentials  of  Anatomy  and 

Manual  of  Practical  Dissection,  .  .  .  18 
Semple — Essentials  of  Pathology  and 

Morbid  Anatomy, 25 

BACTERIOLOGY. 

Ball — Essentials  of  Bacteriology,  ...      6 
Crookshank — A  Text- Book  of  Bacteri- 
ology  lo 

Frothingham — Laboratory  Guide,  .  .  II 
Mallory  and  Wright  —  Pathological 

Technique, 16 

McFarland — Pathogenic  Bacteria,    .    .    17 

CHARTS,  DIET-LISTS,  ETC. 

Griffith — Infant's  Weight  Chart,    ...  12 

Hart — Diet  in  Sickness  and  in  Health,  .  13 

Keen — Operation  Blank, 15 

Laine — Temperature  Chart 15 

Meigs — Feeding  in  Early  Infancy,    .    .  17 

Starr — Diets  for  Infants  and  Children,  .  26 
Thomas — Diet-Lists  and  Sick-Room 

Dietary, 28 

CHEMISTRY  AND  PHYSICS. 

Brockway — Essentials  of  Medical  Phys- 
ics,   ' 7 

Wolff — Essentials  of  Medical  Chemistry,  29 

CHILDREN. 

An  American  Text-Book  of  Diseases 

of  Children, 3 

Griffith — Care  of  the  Baby, 12 

Griffith — Infant's  Weight  Chart,  ...  12 
Meigs — Feeding  in  Early  Infancy,  .  .  17 
Powell — Essentials  of  Dis.  of  Children,  19 
Starr— Diets  for  Infants  and  Children,  .  26 

DIAGNOSIS. 

Cohen  and  Eshner— Essentials  of  Di- 
agnosis  9 

Corwin — Physical  Diagnosis,      ....      9 

Macdonald — Surgical  Diagnosis  and 
Treatment,  16 

Vierordt — Medical  Diagnosis,    ....    29 

DICTIONARIES. 

Dorland— Pocket  Dictionary,     .    .  .  .  10 

Keating — Pronouncing  Dictionary,  .  .  14 

Morten — Nurse's  Dictionary,     .    .  .  .  18 


EYE,  EAR,  NOSE,  AND  THROAT. 

An  American  Text- Book  of  Diseases 

of  the  Eye,  Ear,  Nose,  and  Throat,  .  3 
De  Schweinitz — Diseases  of  the  Eye, .  10 
Gleason — Essentials  of  Dis.  of  the  Ear,  1 1 
Jackson  and  Gleason — Essentials  of 

Diseases  of  the  Eye,  Nose,  and  Throat,  14 
Kyle — Diseases  of  the  Nose  and  Throat,  15 

GENITO-URINARY. 

An  American  Text-Book  of  Genito- 

Urinary  and  Skin  Diseases, 4 

Hyde  and  Montgomery — Syphilis  and 

the  Venereal  Diseases, 13 

Martin — Essentials   of   Minor   Surgery, 

Bandaging,  and  Venereal  Diseases,  .  16 
Saundby — Renal  and  Urinary  Diseases,  24 
Senn — Genito-Urinary  Tuberculosis,  .  25 
Vecki — Sexual  Impotence, 28 

GYNECOLOGY. 

American  Text- Book  of  Gynecology,  4 

Cragin — Essentials  of  Gynecology,    .    .  9 

Garrigues — Diseases  of  Women,  .    .    .  n 

Long — Syllabus  of  Gynecology,     ...  15 

Penrose — Diseases  of  Women,  .    .    .    .  18 

Sutton  and  Giles — Diseases  of  Women,  28 

MATERIA  MEDICA,  PHARMACOL- 
OGY, AND  THERAPEUTICS. 

An  American  Text-Book  of  Applied 

Therapeutics, 3 

Butler— Text-Book  of  Materia  Medica, 

Therapeutics  and  Pharmacology,  ...  8 
Cerna — Notes  on  the  Newer  Remedies,  8 
Griffin — Materia  Med.  and  Therapeutics,  12 
Morris — Essentials  of  Materia  Medica 

and  Therapeutics, 17 

Saunders'  Pocket  Medical  Formulary,  24 
Sayre — Essentials  of  Pharmacy,  ...  24 
Stevens — Essentials  of  Materia  Medica 

and  Therapeutics, 26 

Stoney — Materia  Medica  for  Nurses,  .  28 
Thornton — Dose-Book  and  Manual  of 

Prescription-Writing, .28 

MEDICAL   JURISPRUDENCE   AND 
TOXICOLOGY. 

An  American  Text-Book  of  Legal 
Medicine  and  Toxicology, 4 

Chapman — Medical  Jurisprudence  and 
Toxicology, 8 

Semple — Essentials  of  Legal  Medicine, 
Toxicology,  and  Hygiene, 25 


Medical  Publications  of  W.  B.  Sannders. 


31 


NERVOUS  AND  MENTAL 
DISEASES,  ETC. 

Burr — Nervous  Diseases, 7 

Chapin — Compendium  of  Insanity,  .  .  8 
Church  and  Peterson — Nervous  and 

Mental  Diseases, 8 

Shaw — Essentials  of  Nervous  Diseases 

and  Insanity, 26 

NURSING. 

An  American  Text-Book  of  Nursing,  29 

Griffith— The  Care  of  the  Baby,    ...  12 

Hampton — Nursing,    . 12 

Hart — Diet  in  Sickness  and  in  Health,  13 

Meigs — Feeding  in  Early  Infancy,    .    .  17 

Morten — Nurse's  Dictionary,     .    .    .    .  18 

Stoney — Practical  Points  in  Nursing,    .  27 

OBSTETRICS. 

An  American  Text-Book  of  Obstetrics,  4 
Ashton — Essentials  of  Obstetrics,  ...  6 
Boisliniere — Obstetric  Accidents,  Emer- 
gencies, and  Operations, 7 

Borland — Manual  of  Obstetrics,    .    .    .  lo 

Hirst— Text-Book  of  Obstetrics,    ...  13 

Morris — Syllabus  of  Obstetrics,  .    .    .    .  18 

PATHOLOGY. 

An  American  Text-Book  of  Pathology,  5 
Mallory  and  Wright  —  Pathological 

Technique, 16 

Semple — Essentials   of    Pathology  and 

Morbid  Anatomy, 25 

Senn — Pathology  and  Surgical  Treat- 
ment of  Tumors, ^"25 

Stengel— Text- Book  of  Pathology,   .    .    26 
Warren — Surgical  Pathology  and  Thera- 
peutics,    29 

PHYSIOLOGY. 

An  American  Text-Book  of  Physi- 
ology,   5 

Hare — Essentials  of  Physiology,  ...  13 
Raymond — Manual  of  Physiology,  .  .  19 
Stewart — Manual  of  Physiology,  ...  27 

PRACTICE  OF  MEDICINE. 

An  American  Text-Book  of  the  The- 
ory and  Practice  of  Medicine,  ....  5 

An  American  Year-Book  of  Medicine 
and  Surgery,  6 

Anders — Text-Book  of  the  Practice  of 
Medicine, 6 

Lockwood — Manual  of  the  Practice  of 
Medicine, 15 

Morris — Essentials  of  the  Practice  of 
Medicine, » 17 

Rowland  and  Hedley  —  Archives  of 
the  Roentgen  Ray, 19 

Stevens — Manual  of  the  Practice  of 
Medicine, 27 

SKIN  AND  VENEREAL. 

An  American  Text-Book  of  Genito- 
Urinary  and  Skin  Diseases, 3 


Hyde  and  Montgomery— Syphilis  and 
the  Venereal  Diseases, 13 

Martin — Essentials  of  Minor  Surgery, 
Bandaging,  and  Venereal  Diseases,  .  16 

Pringle— Pictorial  Atlas  of  Skin  Dis- 
eases and  Syphilitic  Affections,  ...  19 

Stelwagon— Essentials  of  Diseases  of 
the  Skin, 26 

SURGERY. 

An  American  Text-Book  of  Surgery,     5 
An  American  Year-Book  of  Medicine 

and  Surgery, 5 

Beck — Manual  of  Surgical  Asepsis,  .    .      7 
DaCosta — Manual  of  Surgery,  .    .    .    .    10 

Keen— Operation  Blank, i$ 

Keen — The  Surgical  Complications  and 

Sequels  of  Typhoid  Fever, 15 

Macdonald — Surgical    Diagnosis    and 

Treatment, 16 

Martin — Essentials   of    Minor  Surgery, 

Bandaging,  and  Venereal  Diseases,    .    16 
Martin — Essentials  of  Surgery, .    .    .    .    16 
Moore — Orthopedic  Surgery,  .       ".    .    .    17 
Pye — Elementary  Bandaging  and  Surgi- 
cal Dressing, 19 

Rowland    and    Hedley— Archives  of 

the  Roentgen  Ray, 19 

Senn — Genito-Urinary  Tuberculosis,     .    25 

Senn  — Syllabus  of  Surgery, 25 

Senn — Pathology  and  Surgical  Treat- 
ment of  Tumors, 25 

Warren — Surgical  Pathology  and  Ther- 
apeutics,   29 

URINE  AND  URINARY  DISEASES. 

Saundby — Renal  and  Urinary  Diseases,  24 
Wolff— Essentials   of    Examination   of 
Urine, 29 


MISCELLANEOUS. 

Bastin — Laboratory   Exercises   in   Bot- 
any,          7 

Gould  and  Pyle — Anomalies  and  Curi- 
osities of  Medicine, n 

Grafstrom — Massage,     .......     12 

Keating — How   to   Examine    for   Life 

Insurance, 14 

Rowland    and    Hedley — Archives   of 

the  Roentgen  Ray, 19 

Saunders'  Medical  Hand-Atlases,  .  .  2 
Saunders'  New  Series  of  Manuals,  22,  23 
Saunders'  Pocket  Medical  Formulary,  .  24 
Saunders'  Question-Compends,  .  .  20,  21 
Senn — Pathology  and  Surgical  Treat- 
ment of  Tumors, 25 

Stewart  and  Lawrance — Essentials  of 

Medical  Electricity, 27 

Thornton — Dose-Book  and  Manual  of 

Prescription-Writing, 28 

Van  Valzah  and  Nisbet— Diseases  of 
the  Stomach, 28 


IN  PRESS 
FOR  PUBLICATION  EARLY  IN  THE  FALL  OF  1899. 


THE   INTERNATIONAL  TEXT-BOOK  OF  SURGERY.     In  two  volumes. 

By  American  and  British  authors.  Edited  by  J.  COLLINS  WARREN,  M.  D.,  LL.D., 
Professor  of  Surgery,  Harvard  Medical  School,  Boston ;  Surgeon  to  the  Massachusetts 
General  Hospital;  and  A.  PEARCE  GOULD,  M.S.,  F.  R.  C.  S.,  Eng.,  Lecturer  on 
Practical  Surgery  and  Teacher  of  Operative  Surgery,  Middlesex  Hospital  Medical 
School;  Surgeon  to  the  Middlesex  Hospital,  London,  England.  Vol.  I.  Handsome 
octavo  volume  of  about  950  pages,  with  over  400  beautiful  illustrations  in  the  text, 
and  9  lithographic  plates. 

HEISLER'S  EMBRYOLOGY. 

A  Text-Book  of  Embryology.  By  JOHN  C.  HEISLER,  M.  D.,  Professor  of 
Anatomy  in  the  Medico-Chirurgical  College,  Philadelphia.  I2mo  volume  of  about 
325  pages,  handsomely  illustrated. 

KYLE  ON  THE  NOSE  AND  THROAT. 

Diseases  of  the  Nose  and  Throat.  By  D.  BRADEN  KYLE,  M.  D.,  Clinical  Pro- 
fessor of  Laryngology  and  Rhinology,  Jefferson  Medical  College,  Philadelphia;  Con- 
sulting Laryngologist,  Rhinologist,  and  Otologist,  St.  Agnes'  Hospital.  Octavo  volume 
of  about  630  pages,  with  over  150  illustrations  and  6  lithographic  plates. 

PRYOR-PELVIC   INFLAMMATIONS. 

The  Treatment  of  Pelvic  Inflammations  through  the  Vagina.     By  W.  R. 

PRYOR,  M.  D.,  Professor  of  Gynecology  in  the  New  York  Polyclinic.  I2mo  volume 
of  about  250  pages,  handsomely  illustrated. 

ABBOTT  ON  TRANSMISSIBLE   DISEASES. 

The  Hygiene  of  Transmissible  Diseases:  their  Causation,  Modes  of 
Dissemination,  and  Methods  of  Prevention.  By  A.  C.  ABBOTT,  M.  !>.,  Pro- 
fessor of  Hygiene  in  the  University  of  Pennsylvania ;  Director  of  the  Laboratory  of 
Hygiene.  Octavo  volume  of  about  325  pages,  containing  a  number  of  charts  and 
maps,  and  numerous  illustrations. 

JACKSON— DISEASES  OF  THE   EYE. 

A  Manual  of  Diseases  of  the  Eye.  By  EDWARD  JACKSON,  A.  M.,  M.  D., 
late  Professor  of  Diseases  of  the  Eye  in  the  Philadelphia  Polyclinic  and  College  for 
Graduates  in  Medicine.  I2mo  volume  of  over  500  pages,  with  about  175  beautiful 
illustrations  from  drawings  by  the  author. 


QR46 
BQ.4 

1898 


McF 
A 


elrlend,    J. 
text-book 

bacterija 


ogenic 


upon 


D7754 
the  path- 
2d  ed. 


Uni 


